Proteasome inhibitors and uses thereof

ABSTRACT

The compounds of the present invention are represented by the following compounds having Formula (I) where the substituents R, R 1 -R 5 , k, m, n, and q are as defined herein. These compounds are used in the treatment of cancer, immunologic disorders, autoimmune disorders, neurodegenerative disorders, or inflammatory disorders, infectious disease, or for providing immunosuppression for transplanted organs or tissues.

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 62/242,139 filed Oct. 15, 2015, which is herebyincorporated by reference in its entirety.

FIELD OF USE

The present invention relates to proteasome inhibitors and uses thereof.

BACKGROUND OF THE INVENTION

Proteasomes are highly conserved self-compartmentalizing proteases foundin three kingdoms of life. A proteasome is a large, ATP-dependent,multi-subunit, barrel-shaped N-terminal nucleophile hydrolase present inthe cytosol and nucleus of eukaryotic cells and is responsible for thedegradation of the majority of cellular proteins (Baumeister et al.,“The Proteasome: Paradigm of a Self-Compartmentalizing Protease,” Cell92(3):367-380 (1998); Goldberg A L., “Functions of the Proteasome: fromProtein Degradation and Immune Surveillance to Cancer Therapy,” Biochem.Soc. Trans. 35(Pt 1): 12-17 (2007)). Through regulated degradation, aproteasome regulates protein homeostasis, the cell cycle, signaltransduction, protein trafficking, immune responses, etc, which areimportant cellular functions. Degradation product oligopeptides arereservoirs of antigenic peptides for MHC class I antigen presentation.

Proteasome inhibition interrupts many cellular pathways, particularly,the NF-kB activation pathway, the induction of unfolded proteinresponse, and ER stress, while strongly inducing apoptosis. For thisreason, highly specific proteasome inhibitors have been approved for thetreatment of hematological cancer. Proteasome inhibitors can alsomarkedly limit the overall supply of peptides for MHC class I moleculesand thus block antigen presentation (Rock et al., “Protein Degradationand the Generation of MHC Class I-Presented Peptides,” Adv. Immunol.80:1-70 (2002)). As a result, proteasome inhibitors reduce immuneresponse via multiple routes.

Plasmodium falciparum: (P. falciparum), the most deadly of the humanmalarias, accounts for nearly 0.5 million deaths a year, primarily inchildren (Zhang et al., “Transcriptome Analysis Reveals Unique MetabolicFeatures in the Cryptosporidium Parvum Oocysts Associated withEnvironmental Survival and Stresses,” BMC Genomics 13:647 (2012)). Themost important current therapies are combinations of artemisinins (ART).The emergence of ART resistant parasites (Ariey et al., “A MolecularMarker of Artemisinin-Resistant Plasmodium Falciparum Malaria,” Nature505(7481):50-55 (2014); Straimer et al., “K13-Propeller Mutations ConferArtemisinin Resistance in Plasmodium Falciparum Clinical Isolates,”Science 347(6220):428-431 (2015); Dogovski et al., “Targeting the CellStress Response of Plasmodium Falciparum to Overcome ArtemisininResistance,” PLoS Biol. 13(4):e1002132 (2015); Mbengue et al., “AMolecular Mechanism of Artemisinin Resistance in Plasmodium FalciparumMalaria,” Nature 520(7549):683-687 (2015)) highlights the need for newantimalarials with novel targets (Wells T N et al., “Malaria Medicines:a Glass Half Full?” Nat. Rev. DrugDiscov. 14(6):424-442 (2015)).Upregulation of the ubiquitin proteasome system (UPS) is important forsurvival of artemisinin-resistant parasites and emphasizes theimportance of the UPS in parasite survival and its importance as a drugtarget moving forward (Dogovski et al., “Targeting the Cell StressResponse of Plasmodium Falciparum to Overcome Artemisinin Resistance,”PLoS Biol. 13(4):e1002132 (2015); Mok et al., “Drug Resistance.Population Transcriptomics of Human Malaria Parasites Reveals theMechanism of Artemisinin Resistance,” Science 347(6220):431-435(2015)).

Proteasome inhibitors are known to kill malaria parasites in vitro andare efficacious against multiple parasite stages; peptide epoxyketoneinhibitors, a peptide vinyl sulfone inhibitor and a cyclic peptideinhibitor, have potent anti-malarial activities (Dogovski et al.,“Targeting the Cell Stress Response of Plasmodium Falciparum to OvercomeArtemisinin Resistance,” PLoS Biol. 13(4):e1002132 (2015); FeatherstoneC. “Proteasome Inhibitors in Development for Malaria,” Mol. Med. Today3(9):367 (1997); Gantt et al., “Proteasome Inhibitors Block Developmentof Plasmodium Spp,” Antimicrob. Agents Chemother. 42(10):2731-2738(1948); Aminake et al., “The Proteasome of Malaria Parasites: AMulti-Stage Drug Target for Chemotherapeutic Intervention?” Int. J.Parasitol. Drugs Drug Resist. 2:1-10 (2012); Li et al., “Validation ofthe Proteasome as a Therapeutic Target in Plasmodium Using anEpoxyketone Inhibitor with Parasite-Specific Toxicity,” Chem. Biol.19(12):1535-1545 (2012); Tschan et al., “Broad-Spectrum AntimalarialActivity of Peptido Sulfonyl Fluorides, a New Class of ProteasomeInhibitors,” Antimicrob. Agents Chemother. 57(8):3576-8354 (2013); Li etal., “Assessing Subunit Dependency of the Plasmodium Proteasome UsingSmall Molecule Inhibitors and Active Site Probes,” ACS Chem. Biol.9(8):1869-1876 (2014); Li et al., “Structure- and Function-Based Designof Plasmodium-Selective Proteasome Inhibitors,” Nature 530(7589):233-236(2016)). Bortezomib (BTZ) and MLN-273 were effective against plasmodiumin blood and liver stages (Lindenthal et al., “The Proteasome InhibitorMLN-273 Blocks Exoerythrocytic and Erythrocytic Development ofPlasmodium Parasites,” Parasitology 131(Pt 1):37-44 (2005); Reynolds etal., “Antimalarial Activity of the Anticancer and Proteasome InhibitorBortezomib and its Analog ZL3B,” BMC. Clin. Pharmacol. 7:13 (2007));MG-132 against blood stage and gametocytes (Lindenthal et al., “TheProteasome Inhibitor MLN-273 Blocks Exoerythrocytic and ErythrocyticDevelopment of Plasmodium Parasites,” Parasitology 131(Pt 1):37-44(2005); Prudhomme et al., “Marine Actinomycetes: a New Source ofCompounds Against the Human Malaria Parasite,” PLoS One 3(6):e2335(2008)); epoxomicin against blood and liver stages and gametocytes(Aminake et al., “Thiostrepton and Derivatives Exhibit Antimalarial AndGametocytocidal Activity by Dually Targeting Parasite Proteasome andApicoplast,” Antimicrob. Agents Chemother. 55(4):1338-1348 (2011);Czesny et al., “The Proteasome Inhibitor Epoxomicin Has PotentPlasmodium Falciparum Gametocytocidal Activity,” Antimicrob. AgentsChemother. 53(10):4080-4085 (2009); Kreidenweiss et al., “ComprehensiveStudy of Proteasome Inhibitors Against Plasmodium Falciparum LaboratoryStrains and Field Isolates From Gabon,” Malar. J. 7:187 (2008); Li etal., “Validation of the Proteasome as a Therapeutic Target in PlasmodiumUsing an Epoxyketone Inhibitor With Parasite-Specific Toxicity,” Chem.Biol. 19(12):1535-1545 (2012)). These inhibitors are in general notspecies selective. They are cytotoxic to host cells and unsuitable fortreating malaria. There is an urgent need to develop Plasmodium spp.proteasome (Pf20S) selective inhibitors that target parasite proteasomesover human host proteasomes.

Degradation of the majority of cytosolic proteins is a highly regulated,ATP-dependent cellular activity executed by the ubiquitin-proteasomesystem (UPS) (Goldberg, A. L. “Functions of the Proteasome: From ProteinDegradation and Immune Surveillance to Cancer Therapy,” Biochem. Soc.Trans., 35:12-17 (2007)). The UPS plays essential roles in diversecellular activities, including cell cycle control, signal transduction,protein homeostasis, and immune surveillance. The 26S proteasome iscomposed of a hydrolytic 20S core and regulators, such as 19S or 11S.The 20S core that is constitutively expressed in most cells (c-20S) is astack of 4 rings of 14 α and β subunits organized in α₁₋₇β₁₋₇β₁₋₇α₁₋₇fashion, where 2 copies of each caspase-like β1, trypsin-like β2, andchymotrypsin-like β5 active subunits are located in the inner β rings(Baumeister, et al., “The Proteasome: Paradigm of aSelf-Compartmentalizing Protease,” Cell 92:367-380 (1998)). Thechymotrypsin-like β5 active subunits of the 20S have been clinicallyvalidated as a target for the treatment of multiple myeloma and certainlymphomas. Bortezomib (BTZ) and carfilzomib (CFZ) are FDA-approved drugsthat represent two classes of covalent proteasome inhibitors: reversiblepeptide boronates and irreversible peptide epoxyketones, respectively(Borissenko et al., “20S Proteasome and its Inhibitors: CrystallographicKnowledge for Drug Development,” Chem. Rev. 107:687-717 (2007); Parlatiet al., Haematol-Hematol. J. 94:148-149 (2009)). Several other classesof proteasome inhibitors have been identified and optimized, such asβ-lactones and peptide sulfonyl fluorides (Huber et al., “Inhibitors forthe Immuno- and Constitutive Proteasome: Current and Future Trends inDrug Development,” Angew. Chem. Int. Ed. Engl. 51(35):8708-8720 (2012));however, the reactive warheads of these classes pose a great challengeto overcome for developing a drug candidate.

Researchers have been focusing on developing noncovalent proteasomeinhibitors for various isoforms of proteasomes, such as Mycobacteriumtuberculosis proteasome (Bryk et al., “Selective Killing ofNonreplicating Mycobacteria,” Cell Host Microbe 3:137-145 (2008); Hu etal., “Structure of the Mycobacterium Tuberculosis Proteasome andMechanism of Inhibition by a Peptidyl Boronate,” Mol. Microbiol.59:1417-1428 (2006); Li et al., “Structural Basis for the Assembly andGate Closure Mechanisms of the Mycobacterium Tuberculosis 20SProteasome,” Embo J. 29:2037-2047 (2010); Lin et al., “N,C-CappedDipeptides With Selectivity for Mycobacterial Proteasome Over HumanProteasomes: Role of S3 and S1 Binding Pockets,” JAm Chem Soc.135:9968-9971 (2013); Lin et al., “Mycobacterium Tuberculosis prcBAGenes Encode a Gated Proteasome With Broad Oligopeptide Specificity,”Mol. Microbiol. 59:1405-1416 (2006); Lin et al., “Fellutamide B is aPotent Inhibitor of the Mycobacterium Tuberculosis Proteasome,” Arch.Biochem. Biophys. 501:214-220 (2010); Lin et al., “Inhibitors Selectivefor Mycobacterial Versus Human Proteasomes,” Nature 461(7264):621-626(2009); Lin et al., “Distinct Specificities of MycobacteriumTuberculosis and Mammalian Proteasomes for N-Acetyl TripeptideSubstrates,” J. Biol. Chem. 283:34423-31 (2008)) and humanimmunoproteasome (i-20S) (Fan et al., “Oxathiazolones SelectivelyInhibit the Human Immunoproteasome over the Constitutive Proteasome,”ACS Med. Chem. Lett. 5:405-410 (2014)). I-20S is expressed in cells ofthe immune system and other cells exposed to cytokines that are elevatedduring immune responses, where the active subunits β1c, β2c and β5c inc-20S are replaced by β1i, β2i and β5i, respectively (Tanaka K “Role ofProteasomes Modified by Interferon-γ in Antigen Processing,” J. Leukoc.Biol. 56:571-575 (1994); Heink et al., “IFN-γ-Induced Immune Adaptationof the Proteasome System is an Accelerated and Transient Response,”Proc. Natl. Acad. Sci. U.S.A. 102:9241-9246 (2005); Kim et al., “A draftmap of the human proteome,” Nature 509:575-581 (2014)). The i-20S servesdiverse functions in the immune system, including the provision ofoligopeptides for antigen presentation, T-cell differentiation andproliferation (Palombella et al., “Role of the Proteasome and NF-kB inStreptococcal Cell Wall-Induced Polyarthritis,” Proc. Natl. Acad. Sci.U.S.A. 95:15671-15676 (1998); Kalim et al., “Immunoproteasome SubunitLMP7 Deficiency and Inhibition Suppresses Th1 and Th17 but EnhancesRegulatory T Cell Differentiation,” J. Immunol. 189:4182-4193 (2012)).Antibody-secreting plasma cells are highly sensitive to proteasomeinhibition and BTZ, which inhibits both c-20S and i-20S, has been usedin renal transplant recipients to prevent antibody-mediated graftrejection (Aull et al., Clin Transpl 495-498 (2009); Raghavan et al.,“Bortezomib in Kidney Transplantation,” J. Transplant. 2010: 698594(2010); Al-Homsi et al., “Effect of Novel Proteasome andImmunoproteasome Inhibitors on Dendritic Cell Maturation, Function, andExpression of IKb and NfKb,” Transpl. Immunol. 29:1-6 (2013); Pai etal., “Treatment of Chronic Graft-Versus-Host Disease with Bortezomib,”Blood 124:1677-1688 (2014)). BTZ was also reported to be efficacious inpatients with refractory systemic lupus erythematosus (Alexander et al.,“The Proteasome Inhibitior Bortezomib Depletes Plasma Cells andAmeliorates Clinical Manifestations of Refractory Systemic LupusErythematosus,” Ann Rheum Dis 74:1474-1478 (2015)). However, BTZ'ssubstantial mechanism-based toxicity requires use of much reduced dosesin the treatment of non-malignant conditions.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a compound of Formula(I):

wherein

R is H or C₁₋₆ alkyl

R¹ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle, wherein alkyl,alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclicheteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl andbi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocyclecan be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from the groupconsisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl,heteroaryl, non-aromatic heterocycle, and non-aromatic heterocyclesubstituted with ═O;

R² is independently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

R³ is selected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl;

R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, and C₃₋₈cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substituted with—CF₃;

R⁵ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

k is 0 or 2;

m is 1 or 2;

n is 1, 2, or 3; and

p is 1 or 2;

or an oxide thereof, a pharmaceutically acceptable salt thereof, asolvate thereof, or a prodrug thereof.

A second aspect of the present invention relates to a method of treatingcancer, immunologic disorders, autoimmune disorders, neurodegenerativedisorders, or inflammatory disorders in a subject or for providingimmunosuppression for transplanted organs or tissues in a subject. Thismethod includes administering to the subject in need thereof a compoundof the Formula (I):

wherein

R is H or C₁₋₆ alkyl;

R¹ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle, wherein alkyl,alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclicheteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl andbi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocyclecan be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from the groupconsisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl,heteroaryl, non-aromatic heterocycle, and non-aromatic heterocyclesubstituted with ═O;

R² is independently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

R³ is selected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl;

R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, and C₃₋₈cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substituted with—CF₃;

R⁵ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

k is 0 or 2;

m is 1 or 2;

n is 1, 2, or 3; and

p is 1 or 2;

or an oxide thereof, a pharmaceutically acceptable salt thereof, asolvate thereof, or a prodrug thereof.

A third aspect of the present invention relates to a method ofinhibiting chymotryptic β5i in a cell or a tissue. This method includesproviding a compound of Formula (I):

wherein

R is H or C₁₋₆ alkyl;

R¹ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle, wherein alkyl,alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclicheteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl andbi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocyclecan be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from the groupconsisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl,heteroaryl, non-aromatic heterocycle, and non-aromatic heterocyclesubstituted with ═O;

R² is independently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

R³ is selected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl;

R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, and C₃₋₈cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substituted with—CF₃;

R⁵ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

k is 0 or 2;

m is 1 or 2;

n is 1, 2, or 3;

p is 1 or 2; and

contacting a cell or tissue with the compound under conditions effectiveto inhibit chymotryptic β5i.

A fourth aspect of the present invention relates to a method of treatinginfectious disease in a subject. This method includes administering tothe subject in need thereof a compound of the Formula (I):

wherein

R is H or C₁₋₆ alkyl;

R¹ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle, wherein alkyl,alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclicheteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl andbi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocyclecan be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from the groupconsisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl,heteroaryl, non-aromatic heterocycle, and non-aromatic heterocyclesubstituted with ═O;

R² is independently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

R³ is selected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl;

R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, and C₃₋₈cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substituted with—CF₃;

R⁵ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

k is 0 or 2;

m is 1 or 2;

n is 1, 2, or 3; and

p is 1 or 2;

or an oxide thereof, a pharmaceutically acceptable salt thereof, asolvate thereof, or a prodrug thereof.

The present application describes that asparagine-ethylenediamine(AsnDEA) can serve as a versatile scaffold for proteasome inhibitors.Kinetic studies of representative compounds showed a noncompetitivemodality of inhibition. Structure-activity relationship studies guidedthe development of potent, non-covalent, reversible, cell-permeableinhibitors with high selectivity for human immunoproteasomes overconstitutive proteasomes. A selective AsnDEA immunoproteasome inhibitoris selectively cytotoxic against tumor cell lines of multiple myelomaand lymphoma over a liver carcinoma cell line, illustrating thepotential of such compounds against multiple myeloma or otherhematological cancers.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1E are graphs showing inhibition modality of PKS21004 againsthuman proteasomes. Washout of c-20S from the preincubated c-20S andPKS21104 to recover the β5c activity (FIG. 1A). Substrate titration ofhu i-20S (FIG. 1B) and c-20S (FIG. 1D) steady state velocities in thepresence of PKS21004 at the concentrations indicated next to each curve.Data as in (FIG. 1B) were plotted double reciprocal in (FIG. 1C), and(FIG. 1D) in (FIG. 1E). Values of Ki and α for PKS21004 were determinedby fitting to an equation for noncompetitive inhibitors: 0.077 μM, 0.28for i-20S, and 0.55 μM, 0.98, respectively.

FIGS. 2A-2B are graphs showing proteasome inhibition by PKS21221 insidethe cells and its cytotoxicity against transformed cell lines. In FIG.2A, Karpas 1106P cells were treated with PKS21221 or HepG2 for 2 hoursat the concentrations indicated prior to incubation with substrate(Ac-ANW)₂R110 for β5i, suc-LLVY-luciferin for β5 in Karpas cells, andsuc-LLVY-luciferin for β5c in HepG2 cells, respectively. IC50s weredetermined to be 0.154 μM and 0.149 μM. FIG. 2B is a graph showingcytotoxicity of PKS21221 against multiple myeloma cell lines MM1.S andRPMI8226, Karpas and HepG2. Values of IC50 of intracellular proteasomeinhibition by PKS21221 and EC50s of cytotoxicity by PKS21221 were listedin Table 3 supra. Data were average of three independent experiments.

FIG. 3 is a graph showing antiplasmodial activity for compoundsPKS21004, PKS21287, and PKS21224 against Plasmodium falciparum: atgametocyte stage. Dihydroartemisinin and methylene blue were used aspositive controls.

FIGS. 4A-4C show that PKS21004 leads to accumulation ofpoly-ubiquitinated proteins in P. falciparum cells (FIG. 4A); PKS21004and PKS21287 block labeling of β5 active subunits of Pf20S by proteasomeactivity probe MV151 (FIG. 4B); and PKS21004 inhibits P. berghei growthin liver HepG2 cells after 6- (square and line) and 14-hours (circle andline) incubation and PKS21004 also prevents invasion of liver cells byP. berghei after removing PKS21004 followed a 2-hour pretreatment(triangle and line) (FIG. 4C).

FIG. 5 is a graph showing that PKS21221 noncompetitively inhibitsimmunoproteasome beta5i activity.

DETAILED DESCRIPTION OF INVENTION

One aspect of the present invention relates to a compound of Formula(I):

wherein

R is H or C₁₋₆ alkyl

R¹ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle, wherein alkyl,alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclicheteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl andbi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocyclecan be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from the groupconsisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl,heteroaryl, non-aromatic heterocycle, and non-aromatic heterocyclesubstituted with ═O;

R² is independently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

R³ is selected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl;

R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, and C₃₋₈cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substituted with—CF₃;

R⁵ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

k is 0 or 2;

m is 1 or 2;

n is 1, 2, or 3; and

p is 1 or 2;

or an oxide thereof, a pharmaceutically acceptable salt thereof, asolvate thereof, or a prodrug thereof.

As used above, and throughout the description herein, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings. If not defined otherwise herein, all technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of ordinary skill in the art to which this technologybelongs. In the event that there is a plurality of definitions for aterm herein, those in this section prevail unless stated otherwise.

The term “alkyl” means an aliphatic hydrocarbon group which may bestraight or branched having about 1 to about 6 carbon atoms in thechain. Branched means that one or more lower alkyl groups such asmethyl, ethyl or propyl are attached to a linear alkyl chain. Exemplaryalkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, n-pentyl, and 3-pentyl.

The term “alkenyl” means an aliphatic hydrocarbon group containing acarbon-carbon double bond and which may be straight or branched havingabout 2 to about 6 carbon atoms in the chain. Particular alkenyl groupshave 2 to about 4 carbon atoms in the chain. Branched means that one ormore lower alkyl groups such as methyl, ethyl, or propyl are attached toa linear alkenyl chain. Exemplary alkenyl groups include ethenyl,propenyl, n-butenyl, and i-butenyl. The term “alkenyl” may also refer toa hydrocarbon chain having 2 to 6 carbons containing at least one doublebond and at least one triple bond.

The term “cycloalkyl” means a non-aromatic mono- or multicyclic ringsystem of about 3 to about 8 carbon atoms, preferably of about 5 toabout 7 carbon atoms. Exemplary monocyclic cycloalkyls includecyclopentyl, cyclohexyl, cycloheptyl, and the like.

The term “aryl” means an aromatic monocyclic or multicyclic ring systemof 6 to about 14 carbon atoms, preferably of 6 to about 10 carbon atoms.Representative aryl groups include phenyl and naphthyl.

As used herein, “biphenyl” or “bi-phenyl” refers to a phenyl groupsubstituted by another phenyl group.

The term “heteroaryl” or “Het” means an aromatic monocyclic ormulticyclic ring system of about 5 to about 14 ring atoms, preferablyabout 5 to about 10 ring atoms, in which one or more of the atoms in thering system is/are element(s) other than carbon, for example, nitrogen,oxygen, or sulfur. In the case of multicyclic ring system, only one ofthe rings needs to be aromatic for the ring system to be defined as“Heteroaryl”. Preferred heteroaryls contain about 5 to 6 ring atoms. Theprefix aza, oxa, thia, or thio before heteroaryl means that at least anitrogen, oxygen, or sulfur atom, respectively, is present as a ringatom. A nitrogen atom of a heteroaryl is optionally oxidized to thecorresponding N-oxide. Representative heteroaryls include pyridyl,2-oxo-pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl,furanyl, pyrrolyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl,thiadiazolyl, tetrazolyl, indolyl, isoindolyl, benzofuranyl,benzothiophenyl, indolinyl, 2-oxoindolinyl, dihydrobenzofuranyl,dihydrobenzothiophenyl, indazolyl, benzimidazolyl, benzooxazolyl,benzothiazolyl, benzoisoxazolyl, benzoisothiazolyl, benzotriazolyl,benzo[1,3]dioxolyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl,pthalazinyl, quinoxalinyl, 2,3-dihydro-benzo[1,4]dioxinyl,benzo[1,2,3]triazinyl, benzo[1,2,4]triazinyl, 4H-chromenyl, indolizinyl,quinolizinyl, 6aH-thieno[2,3-d]imidazolyl, 1H-pyrrolo[2,3-b]pyridinyl,imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl,[1,2,4]triazolo[4,3-a]pyridinyl, [1,2,4]triazolo[1,5-a]pyridinyl,thieno[2,3-b]furanyl, thieno[2,3-b]pyridinyl, thieno[3,2-b]pyridinyl,furo[2,3-b]pyridinyl, furo[3,2-b]pyridinyl, thieno[3,2-d]pyrimidinyl,furo[3,2-d]pyrimidinyl, thieno[2,3-b]pyrazinyl, imidazo[1,2-a]pyrazinyl,5,6,7,8-tetrahydroimidazo[1,2-a]pyrazinyl,6,7-dihydro-4H-pyrazolo[5,1-c][1,4]oxazinyl,2-oxo-2,3-dihydrobenzo[d]oxazolyl, 3,3-dimethyl-2-oxoindolinyl,2-oxo-2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl,benzo[c][1,2,5]oxadiazolyl, benzo[c][1,2,5]thiadiazolyl,3,4-dihydro-2H-benzo[b][1,4]oxazinyl,5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl,[1,2,4]triazolo[4,3-a]pyrazinyl,3-oxo-[1,2,4]triazolo[4,3-a]pyridin-2(3H)-yl, and the like.

As used herein, “biheteroaryl” or “bi-heteroaryl” refers to a heteroarylgroup substituted by another heteroaryl group.

As used herein, “heterocyclyl” or “heterocycle” refers to a stable 3- to18-membered ring (radical) which consists of carbon atoms and from oneto five heteroatoms selected from the group consisting of nitrogen,oxygen and sulfur. For purposes of this application, the heterocycle maybe a monocyclic, or a polycyclic ring system, which may include fused,bridged, or spiro ring systems; and the nitrogen, carbon, or sulfuratoms in the heterocycle may be optionally oxidized; the nitrogen atommay be optionally quaternized; and the ring may be partially or fullysaturated. Examples of such heterocycles include, without limitation,azepinyl, azocanyl, pyranyl dioxanyl, dithianyl, 1,3-dioxolanyl,tetrahydrofuryl, dihydropyrrolidinyl, decahydroisoquinolyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, oxazolidinyl,oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl,pyrazolidinyl, thiazolidinyl, tetrahydropyranyl, thiamorpholinyl,thiamorpholinyl sulfoxide, and thiamorpholinyl sulfone. Furtherheterocycles and heteroaryls are described in Katritzky et al., eds.,Comprehensive Heterocyclic Chemistry: The Structure, Reactions,Synthesis and Use of Heterocyclic Compounds, Vol. 1-8, Pergamon Press,N.Y. (1984), which is hereby incorporated by reference in its entirety.

As used herein, “biheterocyclyl” or “bi-heterocyclyl” refers to aheterocyclyl group substituted by another heterocyclyl or heterocyclegroup.

The term “non-aromatic heterocycle” means a non-aromatic monocyclicsystem containing 3 to 10 atoms, preferably 4 to about 7 carbon atoms,in which one or more of the atoms in the ring system is/are element(s)other than carbon, for example, nitrogen, oxygen, or sulfur.Representative non-aromatic heterocycle groups include pyrrolidinyl,2-oxopyrrolidinyl, piperidinyl, 2-oxopiperidinyl, azepanyl,2-oxoazepanyl, 2-oxooxazolidinyl, morpholino, 3-oxomorpholino,thiomorpholino, 1,1-dioxothiomorpholino, piperazinyl,tetrohydro-2H-oxazinyl, and the like.

The term “monocyclic” used herein indicates a molecular structure havingone ring.

The term “bicyclic” used herein indicates a molecular structure havingtwo ring.

The term “polycyclic” or “multi-cyclic” used herein indicates amolecular structure having two or more rings, including, but not limitedto, fused, bridged, or spiro rings.

Terminology related to “protecting”, “deprotecting,” and “protected”functionalities occurs throughout this application. Such terminology iswell understood by persons of skill in the art and is used in thecontext of processes which involve sequential treatment with a series ofreagents. In that context, a protecting group refers to a group which isused to mask a functionality during a process step in which it wouldotherwise react, but in which reaction is undesirable. The protectinggroup prevents reaction at that step, but may be subsequently removed toexpose the original functionality. The removal or “deprotection” occursafter the completion of the reaction or reactions in which thefunctionality would interfere. Thus, when a sequence of reagents isspecified, as it is in the processes described herein, the person ofordinary skill can readily envision those groups that would be suitableas “protecting groups.” Suitable groups for that purpose are discussedin standard textbooks in the field of chemistry, such as Greene,Protective Groups in Organic Synthesis, John Wiley & Sons, New York(1991), which is hereby incorporated by reference in its entirety.

The term “halo” or “halogen” means fluoro, chloro, bromo, or iodo.

The term “substituted” or “substitution” of an atom means that one ormore hydrogen on the designated atom is replaced with a selection fromthe indicated group, provided that the designated atom's normal valencyis not exceeded.

“Unsubstituted” atoms bear all of the hydrogen atoms dictated by theirvalency. When a substituent is keto (i.e., =0), then two hydrogens onthe atom are replaced. Combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds; a“stable compound” or “stable structure” is meant to be a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

The term “optionally substituted” is used to indicate that a group mayhave a substituent at each substitutable atom of the group (includingmore than one substituent on a single atom), provided that thedesignated atom's normal valency is not exceeded and the identity ofeach substituent is independent of the others. Up to three H atoms ineach residue are replaced with alkyl, halogen, haloalkyl, hydroxy,loweralkoxy, carboxy, carboalkoxy (also referred to as alkoxycarbonyl),carboxamido (also referred to as alkylaminocarbonyl), cyano, carbonyl,nitro, amino, alkylamino, dialkylamino, mercapto, alkylthio, sulfoxide,sulfone, acylamino, amidino, phenyl, benzyl, heteroaryl, phenoxy,benzyloxy, or heteroaryloxy. “Unsubstituted” atoms bear all of thehydrogen atoms dictated by their valency. When a substituent is keto(i.e., =0), then two hydrogens on the atom are replaced. Combinations ofsubstituents and/or variables are permissible only if such combinationsresult in stable compounds; by “stable compound” or “stable structure”is meant a compound that is sufficiently robust to survive isolation toa useful degree of purity from a reaction mixture, and formulation intoan efficacious therapeutic agent.

The term “method of treating” means amelioration or relief from thesymptoms and/or effects associated with the disorders described herein.As used herein, reference to “treatment” of a patient is intended toinclude prophylaxis.

The term “compounds of the invention”, and equivalent expressions, aremeant to embrace compounds of general Formula (I) as hereinbeforedescribed, which expression includes the prodrugs, the pharmaceuticallyacceptable salts, and the solvates, e.g. hydrates, where the context sopermits. Similarly, reference to intermediates, whether or not theythemselves are claimed, is meant to embrace their salts, and solvates,where the context so permits. For the sake of clarity, particularinstances when the context so permits are sometimes indicated in thetext, but these instances are purely illustrative and it is not intendedto exclude other instances when the context so permits.

The term “pharmaceutically acceptable salts” means the relativelynon-toxic, inorganic, and organic acid addition salts, and base additionsalts, of compounds of the present invention. These salts can beprepared in situ during the final isolation and purification of thecompounds. In particular, acid addition salts can be prepared byseparately reacting the purified compound in its free base form with asuitable organic or inorganic acid and isolating the salt thus formed.Exemplary acid addition salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate,oleate, palmitate, stearate, laurate, borate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,naphthylate, mesylate, glucoheptonate, lactiobionate, sulphamates,malonates, salicylates, propionates, methylene-bis-b-hydroxynaphthoates,gentisates, isethionates, di-p-toluoyltartrates, methane-sulphonates,ethanesulphonates, benzenesulphonates, p-toluenesulphonates,cyclohexylsulphamates and quinateslaurylsulphonate salts, and the like(see, for example, Berge et al., “Pharmaceutical Salts,” J. Pharm. Sci.,66:1-9 (1977) and Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Company, Easton, Pa., 1985, p. 1418, which are herebyincorporated by reference in their entirety). Base addition salts canalso be prepared by separately reacting the purified compound in itsacid form with a suitable organic or inorganic base and isolating thesalt thus formed. Base addition salts include pharmaceuticallyacceptable metal and amine salts. Suitable metal salts include thesodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts.The sodium and potassium salts are preferred. Suitable inorganic baseaddition salts are prepared from metal bases which include, for example,sodium hydride, sodium hydroxide, potassium hydroxide, calciumhydroxide, aluminium hydroxide, lithium hydroxide, magnesium hydroxide,and zinc hydroxide. Suitable amine base addition salts are prepared fromamines which have sufficient basicity to form a stable salt, andpreferably include those amines which are frequently used in medicinalchemistry because of their low toxicity and acceptability for medicaluse, such as ammonia, ethylenediamine, N-methyl-glucamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethyl enediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, triethylarnine, dibenzylamine,ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, ethylamine, basic amino acids, e.g., lysine andarginine, dicyclohexylamine, and the like.

The term “pharmaceutically acceptable prodrugs” as used herein meansthose prodrugs of the compounds useful according to the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “prodrug” means compoundsthat are rapidly transformed in vivo to yield the parent compound of theabove formula, for example by hydrolysis in blood. Functional groupswhich may be rapidly transformed, by metabolic cleavage, in vivo form aclass of groups reactive with the carboxyl group of the compounds ofthis invention. They include, but are not limited to, such groups asalkanoyl (such as acetyl, propionyl, butyryl, and the like),unsubstituted and substituted aroyl (such as benzoyl and substitutedbenzoyl), alkoxycarbonyl (such as ethoxycarbonyl), trialkylsilyl (suchas trimethyl- and triethysilyl), monoesters formed with dicarboxylicacids (such as succinyl), and the like. Because of the ease with whichthe metabolically cleavable groups of the compounds useful according tothis invention are cleaved in vivo, the compounds bearing such groupsact as pro-drugs. The compounds bearing the metabolically cleavablegroups have the advantage that they may exhibit improved bioavailabilityas a result of enhanced solubility and/or rate of absorption conferredupon the parent compound by virtue of the presence of the metabolicallycleavable group. A thorough discussion of prodrugs is provided in thefollowing: Design of Prodrugs, H. Bundgaard, ed., Elsevier (1985);Methods in Enzymology, K. Widder et al, Ed., Academic Press, 42,p.309-396 (1985); A Textbook of Drug Design and Development,Krogsgaard-Larsen and H. Bundgaard, ed., Chapter 5; “Design andApplications of Prodrugs” p.113-1⁹¹ (1991); Advanced Drug DeliveryReviews, H. Bundgard, 8, p.1-38 (1992); J. Pharm. Sci., 77:285 (1988);Nakeya et al, Chem. Pharm. Bull., 32:692 (1984); Higuchi et al.,“Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S. SymposiumSeries, and Bioreversible Carriers in Drug Design, Edward B. Roche, ed.,American Pharmaceutical Association and Pergamon Press (1987), which areincorporated herein by reference in their entirety. Examples of prodrugsinclude, but are not limited to, acetate, formate, and benzoatederivatives of alcohol and amine functional groups in the compounds ofthe invention.

The term “solvate” refers to a compound of Formula I in the solid state,wherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent for therapeutic administration isphysiologically tolerable at the dosage administered. Examples ofsuitable solvents for therapeutic administration are ethanol and water.When water is the solvent, the solvate is referred to as a hydrate. Ingeneral, solvates are formed by dissolving the compound in theappropriate solvent and isolating the solvate by cooling or using anantisolvent. The solvate is typically dried or azeotroped under ambientconditions.

The term “therapeutically effective amounts” is meant to describe anamount of compound of the present invention effective in inhibiting theproteasome or immunoproteasome and thus producing the desiredtherapeutic effect. Such amounts generally vary according to a number offactors well within the purview of ordinarily skilled artisans given thedescription provided herein to determine and account for. These include,without limitation: the particular subject, as well as its age, weight,height, general physical condition, and medical history; the particularcompound used, as well as the carrier in which it is formulated and theroute of administration selected for it; and, the nature and severity ofthe condition being treated.

The term “pharmaceutical composition” means a composition comprising acompound of Formula (I) and at least one component comprisingpharmaceutically acceptable carriers, diluents, adjuvants, excipients,or vehicles, such as preserving agents, fillers, disintegrating agents,wetting agents, emulsifying agents, suspending agents, sweeteningagents, flavoring agents, perfuming agents, antibacterial agents,antifingal agents, lubricating agents and dispensing agents, dependingon the nature of the mode of administration and dosage forms. Examplesof suspending agents include ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monosterate andgelatin. Examples of suitable carriers, diluents, solvents, or vehiclesinclude water, ethanol, polyols, suitable mixtures thereof, vegetableoils (such as olive oil), and injectable organic esters such as ethyloleate. Examples of excipients include lactose, milk sugar, sodiumcitrate, calcium carbonate, and dicalcium phosphate. Examples ofdisintegrating agents include starch, alginic acids, and certain complexsilicates. Examples of lubricants include magnesium stearate, sodiumlauryl sulphate, talc, as well as high molecular weight polyethyleneglycols.

The term “pharmaceutically acceptable” means it is, within the scope ofsound medical judgement, suitable for use in contact with the cells ofhumans and lower animals without undue toxicity, irritation, allergicresponse and the like, and are commensurate with a reasonablebenefit/risk ratio.

The term “pharmaceutically acceptable dosage forms” means dosage formsof the compound of the invention, and includes, for example, tablets,dragees, powders, elixirs, syrups, liquid preparations, includingsuspensions, sprays, inhalants tablets, lozenges, emulsions, solutions,granules, capsules, and suppositories, as well as liquid preparationsfor injections, including liposome preparations. Techniques andformulations generally may be found in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., latest edition.

Compounds described herein may contain one or more asymmetric centersand may thus give rise to enantiomers, diastereomers, and otherstereoisomeric forms. Each chiral center may be defined, in terms ofabsolute stereochemistry, as (R)- or (S)-. This technology is meant toinclude all such possible isomers, as well as mixtures thereof,including racemic and optically pure forms. Optically active (R)- and(S)-, (−)- and (+)-, or (D)- and (L)-isomers may be prepared usingchiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic doublebonds or other centers of geometric asymmetry, and unless specifiedotherwise, it is intended that the compounds include both E and Zgeometric isomers. Likewise, all tautomeric forms are also intended tobe included.

This technology also envisions the “quaternization” of any basicnitrogen-containing groups of the compounds disclosed herein. The basicnitrogen can be quaternized with any agents known to those of ordinaryskill in the art including, for example, lower alkyl halides, such asmethyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkylsulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aralkyl halides including benzyl and phenethylbromides. Water or oil-soluble or dispersible products may be obtainedby such quaternization.

In the characterization of some of the substituents, it is recited thatcertain substituents may combine to form rings. Unless stated otherwise,it is intended that such rings may exhibit various degrees ofunsaturation (from fully saturated to fully unsaturated), may includeheteroatoms and may be substituted with lower alkyl or alkoxy.

Compounds of Formula (I) can be produced according to known methods. Forexample, compounds of Formula (I) can be prepared according to Scheme 1outlined below.

Coupling of the amine (1) with the carboxylic acid (2) leads toformation of the compound (3). The coupling reaction can be carried outin a variety of solvents, for example in methylene chloride (CH₂Cl₂),tetrahydrofuran (THFE), dimethylformamide (DMF), or other such solventsor in the mixture of such solvents. During the coupling process, thenon-participating carboxylic acids or amines on the reacting set ofamino acids or peptide fragments can be protected by a suitableprotecting group which can be selectively removed at a later time ifdesired. A detailed description of these groups and their selection andchemistry is contained in “The Peptides, Vol, 3”, Gross and Meinenhofer,Eds., Academic Press, New York, 1981, which is hereby incorporated byreference in its entirety. Thus, useful protective groups for the aminogroup are benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (t-BOC),2,2,2-trichl oroethoxycarbonyl (Troc), t-amyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-(trichlorosilyl)ethoxycarbonyl,9-fluorenylmethoxycarbonyl (Fmoc), phthaloyl, acetyl (Ac), formyl,trifluoroacetyl, and the like.

Amine (1) can be prepared according to the general scheme outlined below(Scheme 2).

Amine (5) can be prepared by deprotection of compound (4). Coupling ofthe amine (5) with the carboxylic acid (6) leads to formation of thecompound (7). The coupling reactions are conducted in solvents such asmethylene chloride (CH₂Cl₂), tetrahydrofuran (THF), dimethylformamide(DMF), or other such solvents. During the coupling process, thenon-participating carboxylic acids or amines on the reacting set ofamino acids or peptide fragments can be protected by a suitableprotecting group which can be selectively removed at a later time ifdesired. A detailed description of these groups and their selection andchemistry is contained in “The Peptides, Vol. 3”, Gross and Meinenhofer,Eds., Academic Press, New York, 1981, which is hereby incorporated byreference in its entirety. Thus, useful protective groups for the aminogroup are benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (t-BOC),2,2,2-trichloroethoxycarbonyl (Troc), t-amyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-(trichlorosilyl)ethoxycarbonyl,9-fluorenylmethoxycarbonyl (Fmoc), phthaloyl, acetyl (Ac), formyl,trifluoroacetyl, and the like. Following the deprotection reaction,compound (8) is coupled with amine (9) to form compound (10). Amine (1)can be prepared by deprotection of compound (10).

In one embodiment, compound has the Formula (Ia):

In another embodiment, compound has the Formula (Ib):

In yet another embodiment, compound has the Formula (Ic):

Another embodiment relates to the compound of Formula (I) where alkyl isC₁₋₆ alkyl.

Yet another embodiment relates to the compound of Formula (I) wherealkenyl is C₂₋₆ alkenyl.

Another embodiment relates to the compound of Formula (I) where R¹ isselected from the group consisting of

Yet another embodiment relates to the compound of Formula (I) where R²is selected from the group consisting of H, CH₃,

Another embodiment relates to the compound of Formula (I) where R³ isselected from the group consisting of H,

and R is C₁₋₆ alkyl.

Another embodiment relates to the compound of Formula (I) where thecompound has a structure selected from the group consisting of:

A second aspect of the present invention relates to a method of treatingcancer, immunologic disorders, autoimmune disorders, neurodegenerativedisorders, or inflammatory disorders in a subject or for providingimmunosuppression for transplanted organs or tissues in a subject. Thismethod includes administering to the subject in need thereof a compoundof the Formula (I):

wherein

R is H or C₁₋₆ alkyl;

R¹ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle, wherein alkyl,alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclicheteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl andbi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocyclecan be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from the groupconsisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl,heteroaryl, non-aromatic heterocycle, and non-aromatic heterocyclesubstituted with ═O;

R² is independently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

R³ is selected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl;

R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, and C₃₋₈cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substituted with—CF₃;

R⁵ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

k is 0 or 2;

m is 1 or 2;

n is 1, 2, or 3; and

p is 1 or 2;

or an oxide thereof, a pharmaceutically acceptable salt thereof, asolvate thereof, or a prodrug thereof.

The different forms of Formula (I) discussed above are all applicable tothis embodiment of the present invention.

In one embodiment, an autoimmune disorder is treated. The autoimmunedisorder is selected from the group consisting of arthritis, colitis,multiple sclerosis, lupus, systemic sclerosis, and sjögren syndrome.

In another embodiment, immunosuppression is provided for transplantedorgans or tissues. The immunosuppression is used to prevent transplantrejection and graft-verse-host disease.

In another embodiment, an inflammatory disorder is treated. Theinflammatory disorder is Crohn's disease or ulcerative colitis.

In yet another embodiment, cancer is treated. The cancer is selectedfrom the group consisting of multiple myeloma, lymphoma, and otherhematological cancers.

While it may be possible for compounds of Formula (I) to be administeredas raw chemicals, it will often be preferable to present them as a partof a pharmaceutical composition. Accordingly, another aspect of thepresent invention is a pharmaceutical composition containing atherapeutically effective amount of the compound of Formula (I), or apharmaceutically acceptable salt or solvate thereof, and apharmaceutically acceptable carrier. The carrier must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

In practicing this method of the present invention, agents suitable fortreating a subject can be administered using any method standard in theart. The agents, in their appropriate delivery form, can be administeredorally, intradermally, intramuscularly, intraperitoneally,intravenously, subcutaneously, or intranasally. The compositions of thepresent invention may be administered alone or with suitablepharmaceutical carriers, and can be in solid or liquid form, such astablets, capsules, powders, solutions, suspensions, or emulsions.

The agents of the present invention may be orally administered, forexample, with an inert diluent, or with an assimilable edible carrier,or it may be enclosed in hard or soft shell capsules, or it may becompressed into tablets, or they may be incorporated directly with thefood of the diet. Agents of the present invention may also beadministered in a time release manner incorporated within such devicesas time-release capsules or nanotubes. Such devices afford flexibilityrelative to time and dosage. For oral therapeutic administration, theagents of the present invention may be incorporated with excipients andused in the form of tablets, capsules, elixirs, suspensions, syrups, andthe like. Such compositions and preparations should contain at least0.1% of the agent, although lower concentrations may be effective andindeed optimal. The percentage of the agent in these compositions may,of course, be varied and may conveniently be between about 2% to about60% of the weight of the unit. The amount of an agent of the presentinvention in such therapeutically useful compositions is such that asuitable dosage will be obtained.

Also specifically contemplated are oral dosage forms of the agents ofthe present invention. The agents may be chemically modified so thatoral delivery of the derivative is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe component molecule itself, where said moiety permits (a) inhibitionof proteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecomponent or components and increase in circulation time in the body.Examples of such moieties include: polyethylene glycol, copolymers ofethylene glycol and propylene glycol, carboxymethyl cellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. (Abuchowskiand Davis, “Soluble Polymer-Enzyme Adducts,” In: Enzymes as Drugs,Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp.367-383 (1981), which are hereby incorporated by reference in theirentirety). Other polymers that could be used are poly-1,3-dioxolane andpoly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicatedabove, are polyethylene glycol moieties.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, sucrulose, or saccharin. Whenthe dosage unit form is a capsule, it may contain, in addition to theabove types of materials, a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor.

The agents of the present invention may also be administeredparenterally. Solutions or suspensions of the agent can be prepared inwater suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof in oils. Illustrative oils are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, or mineral oil. In general, water, saline, aqueousdextrose and related sugar solution, and glycols, such as propyleneglycol or polyethylene glycol, are preferred liquid carriers,particularly for injectable solutions. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

When it is desirable to deliver the agents of the present inventionsystemically, they may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Intraperitoneal or intrathecal administration of the agents of thepresent invention can also be achieved using infusion pump devices suchas those described by Medtronic, Northridge, Calif. Such devices allowcontinuous infusion of desired compounds avoiding multiple injectionsand multiple manipulations.

In addition to the formulations described previously, the agents mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The agents of the present invention may also be administered directly tothe airways in the form of an aerosol. For use as aerosols, the agent ofthe present invention in solution or suspension may be packaged in apressurized aerosol container together with suitable propellants, forexample, hydrocarbon propellants like propane, butane, or isobutane withconventional adjuvants. The agent of the present invention also may beadministered in a non-pressurized form such as in a nebulizer oratomizer.

Effective doses of the compositions of the present invention, for thetreatment of cancer or pathogen infection vary depending upon manydifferent factors, including type and stage of cancer or the type ofpathogen infection, means of administration, target site, physiologicalstate of the patient, other medications or therapies administered, andphysical state of the patient relative to other medical complications.Treatment dosages need to be titrated to optimize safety and efficacy.

The percentage of active ingredient in the compositions of the presentinvention may be varied, it being necessary that it should constitute aproportion such that a suitable dosage shall be obtained. Obviously,several unit dosage forms may be administered at about the same time.The dose employed will be determined by the physician and depends uponthe desired therapeutic effect, the route of administration and theduration of the treatment, and the condition of the patient. In theadult, the doses are generally from about 0.01 to about 100 mg/kg bodyweight, preferably about 0.01 to about 10 mg/kg body weight per day byinhalation, from about 0.01 to about 100 mg/kg body weight, preferably0.1 to 70 mg/kg body weight, more especially 0.1 to 10 mg/kg body weightper day by oral administration, and from about 0.01 to about 50 mg/kgbody weight, preferably 0.01 to 10 mg/kg body weight per day byintravenous administration. In each particular case, the doses will bedetermined in accordance with the factors distinctive to the subject tobe treated, such as age, weight, general state of health, and othercharacteristics which can influence the efficacy of the medicinalproduct.

The products according to the present invention may be administered asfrequently as necessary in order to obtain the desired therapeuticeffect. Some patients may respond rapidly to a higher or lower dose andmay find much weaker maintenance doses adequate. For other patients, itmay be necessary to have long-term treatments at the rate of 1 to 4doses per day, in accordance with the physiological requirements of eachparticular patient. Generally, the active product may be administeredorally 1 to 4 times per day. It goes without saying that, for otherpatients, it will be necessary to prescribe not more than one or twodoses per day.

A third aspect of the present invention relates to a method ofinhibiting chymotryptic β5i in a cell or a tissue. This method includesproviding a compound of Formula (I):

wherein

R is H or C₁₋₆ alkyl;

R¹ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle, wherein alkyl,alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclicheteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl andbi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocyclecan be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from the groupconsisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl,heteroaryl, non-aromatic heterocycle, and non-aromatic heterocyclesubstituted with ═O;

R² is independently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

R³ is selected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁-6alkyl;

R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, and C₃₋₈cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substituted with—CF₃;

R⁵ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

k is 0 or 2;

m is 1 or 2;

n is 1, 2, or 3;

p is 1 or 2; and

contacting a cell or tissue with the compound under conditions effectiveto inhibit chymotryptic β5i.

The different forms of Formula (I) discussed above are all applicable tothis embodiment of the present invention.

In one embodiment, the chymotryptic 35i is inhibited selectively over35c.

In another embodiment, the chymotryptic 35c is inhibited selectivelyover 35i.

A fourth aspect of the present invention relates to a method of treatinginfectious disease in a subject. This method includes administering tothe subject in need thereof a compound of the Formula (I):

wherein

R is H or C₁₋₆ alkyl;

R¹ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle, wherein alkyl,alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic and bicyclicheteroaryl and bi-heteroaryl, monocyclic and bicyclic heterocyclyl andbi-heterocyclyl, and monocyclic and bicyclic non-aromatic heterocyclecan be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from the groupconsisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl,heteroaryl, non-aromatic heterocycle, and non-aromatic heterocyclesubstituted with ═O;

R² is independently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

R³ is selected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl;

R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, and C₃₋₈cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substituted with—CF₃;

R⁵ is selected from the group consisting of alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl;

k is 0 or 2;

m is 1 or 2;

n is 1, 2, or 3; and

p is 1 or 2;

or an oxide thereof, a pharmaceutically acceptable salt thereof, asolvate thereof, or a prodrug thereof.

The different forms of Formula (I) discussed above are all applicable tothis embodiment of the present invention.

Likewise, the modes of formulation and administration of the compoundsof Formula (I) discussed above can be used in carrying out this aspectof the present invention.

In one embodiment, the infectious disease is caused by bacterial, viral,parasitic, and fungal infectious agents.

In one embodiment, the infectious disease is caused by a bacteriaselected from the group consisting of Escherichia coli, Salmonella,Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Mycobacteriumtuberculosis, Mycobacterium avium-intracellulare, Yersinia, Francisella,Pasteurella, Brucella, Clostridia, Bordetella pertussis, Bacteroides,Staphylococcus aureus, Streptococcus pneumonia, B-Hemolytic strep.,Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseriagonorrhea, Neisseria meningitides, Hemophilus influenza,Enterococcusfaecalis, Proteus vulgaris, Proteus mirabilis, Helicobacterpylori, Treponema palladium, Borrelia burgdorferi, Borrelia recurrentis,Rickettsial pathogens, Nocardia, and Actinomycetes.

In another embodiment, the infectious disease is caused by a fungalinfectious agent selected from the group consisting of Cryptococcusneoformans, Blastomyces dermatitidis, Histoplasma capsulatum,Coccidioides immitis, Paracoccicioides brasiliensis, Candida albicans,Aspergillusfumigautus, Phycomycetes (Rhizopus), Sporothrix schenckii,Chromomycosis, and Maduromycosis.

In another embodiment, the infectious disease is caused by a viralinfectious agent selected from the group consisting of humanimmunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitisviruses, Epstein-Barr Virus, cytomegalovirus, human papillomaviruses,orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses,rhabdo viruses, polio viruses, toga viruses, bunya viruses, arenaviruses, rubella viruses, and reo viruses.

In yet another embodiment, the infectious disease is caused by aparasitic infectious agent selected from the group consisting ofPlasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodiumovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosomaspp., Entamoeba histolytica, Cryptosporidium, Giardia spp., Trichimonasspp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobiusvermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculusmedinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystiscarinii, and Necator americanis.

In one embodiment, the infectious disease is malaria.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1—Chemicals and Spectroscopy

Unless otherwise stated, all commercially available materials werepurchased from Bachem, Aldrich, P3 BioSystems, or other vendors and wereused as received. All non-aqueous reactions were performed under argonin oven-dried glassware. Routine monitoring of reactions was performedusing Waters Acquity Ultra Performance Liquid Chromatography (UPLC). AllHPLC purifications were done by Varian PrepStar HPLC system or WatersAutopure (mass directed purification system) using Prep C18 5 μm OBD(19×150 mm) column. ¹H- and ¹³C-NMR spectra were acquired on a BrukerDRX-500 spectrometer. Chemical shifts 6 are expressed in parts permillion, with the solvent resonance as an internal standard(chloroform-d, ¹H: 7.26; ¹³C: 77.16 ppm; DMSO-d6, ¹H: 2.50 ppm; ¹³C:39.52 ppm). Hexafluorobenzene was used as internal standard for ¹⁹F NMR.NMR data are reported as following: chemical shift, multiplicity(s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad),coupling constant, and integration.

Example 2—General Procedure for HATU Mediated Amide Bond Formation

To a solution of carboxylic acid (1 equivalent),O-(7-Azabenzotriazole-1-yl)-N,N,N,N′-tetramethyluroniumhexafluorophosphate (HATU, 1.2 equivalent), and1-hydroxy-7-azabenzotriazole (HOAt; 0.6 M in DMF) in DMF, Hunig base(3-5 equiv) was added dropwise at 0° C. The mixture was stirred at 0° C.for 5 minutes and amine (1 equivalent) was added. The reaction mixturewas stirred at 0° C. until complete consumption of starting material(monitored by LCMS). After completion of reaction, water was added tothe reaction mixture and stirred for 30 minutes. The product wasisolated by either filtration or ethyl acetate extraction.

Example 3—General Procedure for Boc-Deprotection

The solution of substrate in dichloromethane was cooled to 0° C.Trifluoroacetic acid (20% v/v with respect to dichloromethane) was addedto the solution drop wise at 0° C. with constant stirring. The mixturewas allowed to warm to room temperature slowly (over a period of 1hour), and stirred until the completion of reaction (monitored by LCMS).Excess trifluoroacetic acid and dichloromethane were evaporated andcrude was dried under vacuum.

Example 4—General Procedure for O-Debenzylation

Palladium on carbon (10%) was added carefully to a solution of substratein methanol. Residual air from the flask was removed and flushed withhydrogen. The mixture was stirred at room temperature for 3-4 hoursunder hydrogen atmosphere using a hydrogen balloon. After completion ofthe reaction, the mixture was filtered through celite. Filtrate wasevaporated and dried under vacuum to give product.

Example 5—General Procedure for N-sulfonamide Synthesis of Amines

Triethylamine (2.0-3.0 eq.) was added to a solution of substrate (amine,generally TFA salt) in dichloromethane at 0° C. The mixture was warmedto room temperature (25° C.) and sulfonyl chloride (1.5 eq.) was addedin one portion. After completion of the reaction (2-3 hours),dichloromethane was evaporated and crude product was isolated by ethylacetate extraction.

Example 6—Synthesis of PKS3070

The title compound was synthesized by following the general procedure ofHATU mediated coupling ofN-tert-butyl-N²-(1-oxo-3-phenylpropyl)-L-Aspargine (778 mg, 2.43 mmol)and N-Boc-ethylenediamine (428 mg, 2.67 mmol). After completion of thereaction, water was added. White precipitate formed, was filtered anddried in air to give product (955 mg, 85%) as a white solid. Product wasused in next step without further purification. ¹H NMR (500 MHz,Chloroform-d) δ 7.32-7.27 (m, 3H), 7.24-7.18 (m, 3H), 6.92 (br, 1H),5.72 (br, 1H), 5.01 (br, 1H), 4.65-4.56 (m, 1H), 3.29-3.14 (m, 4H),3.05-2.92 (m, 2H), 2.70 (dd, J=15.0, 3.7 Hz, 1H), 2.60 (t, J=7.6 Hz,2H), 2.31 (dd, J=15.0, 6.1 Hz, 1H), 1.44 (s, 9H), 1.31 (s, 9H).

Example 7—Synthesis of PKS3072

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS3070 (953 mg, 2.06 mmol). After completion ofthe reaction, excess trifluoroacetic acid and dichloromethane wereevaporated. Crude was dried and triturated with diethyl ether to give awhite solid. Diethyl ether was decanted and white solid was dried undervacuum to give product (980 mg, quant.). Product was used in next stepwithout further purification. ¹H NMR (500 MHz, DMSO-d₆) δ 8.08 (d, J=7.9Hz, 1H), 8.05 (t, J=5.6 Hz, 1H), 7.80 (br, 3H), 7.56 (s, 1H), 7.30-7.23(m, 2H), 7.22-7.14 (m, 3H), 4.50-4.42 (m, 1H), 3.42-3.31 (m, 1H),3.29-3.19 (m, 1H), 2.92-2.82 (m, 2H), 2.80 (t, J=7.9 Hz, 2H), 2.48-2.34(m, 4H), 1.22 (s, 9H).

Example 8—Synthesis of PKS3080

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 1-naphthoic acid (20.7 mg, 0.12 mmol) andPKS3072 (47.6 mg, 0.1 mmol). After completion of the reaction, themixture was purified by HPLC to give product (30.2 mg, 58%) as a whitesolid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.47 (t, J=5.6 Hz, 1H), 8.25-8.18 (m,1H), 8.03-7.92 (m, 4H), 7.66 (dd, J=7.1, 1.3 Hz, 1H), 7.60-7.48 (m, 3H),7.38 (s, 1H), 7.28-7.22 (m, 2H), 7.19-7.12 (m, 3H), 4.57-4.46 (m, 1H),3.45-3.37 (m, 2H), 3.32-3.21 (m, 2H), 2.77 (t, J=8.0 Hz, 2H), 2.46 (dd,J=14.6, 6.0 Hz, 1H), 2.43-2.37 (m, 2H), 2.32 (dd, J=14.6, 7.9 Hz, 1H),1.19 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 171.3, 171.2, 168.9, 168.7,141.3, 134.7, 133.1, 129.8, 129.7, 128.3, 128.1, 128.1, 126.7, 126.2,125.8, 125.5, 125.3, 124.9, 50.2, 50.0, 38.9, 38.7, 38.7, 36.9, 30.9,28.4. HRMS calc. for C₃₀H₃₆N₄O₄Na [M+Na]⁺: 539.2634. Found: 539.2637.

Example 9—Synthesis of PKS21003

The title compound was synthesized by HATU mediated coupling of4-phenylbenzoic acid (104.0 mg, 524.7 μmol) and PKS3072 (250.0 mg, 524.7μmol). After completion of the reaction (1 hour), water was added. Thewhite precipitate obtained was filtered, washed with water and dried inair to give 284.0 mg white solid. The white solid was triturated withethyl acetate and isolated by centrifugation (4700 rpm, 10 min).Isolated white solid (235 mg, 82%) was pure product (by LCMS & NMR). ¹HNMR (500 MHz, DMSO-d₆) δ 8.51-8.44 (m, 1H), 8.00 (d, J=7.9 Hz, 1H),7.99-7.89 (m, 3H), 7.76-7.72 (m, 2H), 7.72-7.67 (m, 2H), 7.52-7.45 (m,2H), 7.43-7.36 (m, 2H), 7.28-7.22 (m, 2H), 7.20-7.12 (m, 3H), 4.54-4.45(m, 1H), 3.36-3.33 (m, 2H), 3.29-3.14 (m, 2-H), 2.78 (t, J=8.0 Hz, 2H),2.48-2.38 (m, 3H), 2.32 (dd, J=15.0, 8.0 Hz, 1H), 1.21 (s, 9H); ¹³C NMR(126 MHz, DMSO-d₆) δ 171.3, 171.2, 168.9, 166.1, 142.7, 141.3, 139.2,133.3, 129.0, 128.3, 128.1, 128.0, 127.9, 126.8, 126.4, 125.9, 50.2,50.1, 38.9, 38.7, 38.6, 36.9, 31.0, 28.4. HRMS calc. for C₃₂H₃₈N₄O₄Na[M+Na]⁺: 565.2791. Found: 565.2786.

Example 10—Synthesis of PKS21004

The title compound was synthesized by HATU mediated coupling of3-phenylbenzoic acid (396.8 mg, 2.00 mol) and PKS3072 (867.2 mg, 1.82mmol). After completion of the reaction (2 hours), water was added tothe reaction mixture. An off white precipitate appeared. The precipitatewas filtered and recrystallized from ethanol to give pure product as awhite solid (905 mg, 92%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.57 (t, J=5.5Hz, 1H), 8.13 (s, 1H), 8.01-7.93 (m, 2H), 7.87-7.79 (m, 2H), 7.76-7.70(m, 2H), 7.54 (t, J=7.7 Hz, 1H), 7.51-7.46 (m, 2H), 7.43-7.35 (m, 2H),7.28-7.22 (m, 2H), 7.19-7.13 (m, 3H), 4.54-4.46 (m, 1H), 3.38-3.15 (m,4H), 2.77 (t, J=7.9 Hz, 2H), 2.48-2.36 (m, 3H), 2.32 (dd, J=14.7, 7.9Hz, 1H), 1.20 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ 171.3, 171.2, 168.9,166.3, 141.3, 140.1, 139.6, 135.1, 129.3, 129.0, 129.0, 128.3, 128.1,127.7, 126.8, 126.4, 125.8, 125.4, 50.2, 50.0, 38.9, 38.7, 38.6, 36.9,30.9, 28.4. HRMS calc. for C₃₂H₃₈N₄O₄Na [M+Na]⁺: 565.2791. Found:565.2774.

Example 11—Synthesis of PKS21025

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-naphthoic acid (5.2 mg, 30 μmol) andPKS3072 (11.9 mg, 25 μmol). After completion of the reaction (1 hour),the mixture was purified by HPLC to give product (11.5 mg, 89%) as awhite solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.61 (t, J=5.6 Hz, 1H), 8.43(s, 1H), 8.03-7.95 (m, 5H), 7.92 (d, J=8.6 Hz, 1H), 7.65-7.53 (m, 2H),7.40 (s, 1H), 7.29-7.20 (m, 2H), 7.20-7.10 (m, 3H), 4.55-4.46 (m, 1H),3.44-3.34 (m, 2H), 3.32-3.18 (m, 2H), 2.77 (t, J=8.0 Hz, 2H), 2.49-2.37(m, 3H), 2.32 (dd, J=14.6, 7.9 Hz, 1H), 1.19 (s, 9H); ¹³C NMR (126 MHz,DMSO-d₆) δ 171.6, 171.6, 169.1, 166.9, 141.4, 134.3, 132.3, 131.9,129.0, 128.5, 128.3, 128.0, 127.8, 127.8, 127.6, 126.9, 126.1, 124.3,50.4, 50.3, 39.2, 38.9, 38.8, 37.1, 31.1, 28.6. HRMS calc. forC₃₀H₃₆N₄O₄Na [M+Na]⁺: 539.2634. Found: 539.2617.

Example 12—Synthesis of PKS21026

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-(4-fluorophenyl)benzoic acid (6.49 mg,30 μmol) and PKS3072 (11.9 mg, 25 μmol). After completion of thereaction (1 hour), the mixture was purified by HPLC to give product(11.0 mg, 78%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.57 (t,J=5.6 Hz, 1H), 8.12-8.07 (m, 1H), 7.99 (d, J=8.0 Hz, 1H), 7.96 (t, J=5.7Hz, 1H), 7.85-7.72 (m, 4H), 7.57-7.50 (m, 1H), 7.39 (s, 1H), 7.35-7.26(m, 2H), 7.28-7.21 (m, 2H), 7.19-7.13 (m, 3H), 4.49 (td, J=7.9, 5.9 Hz,1H), 3.37-3.14 (m, 4H), 2.76 (t, J=8.0 Hz, 2H), 2.49-2.35 (m, 3H), 2.32(dd, J=14.6, 7.9 Hz, 1H), 1.19 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ171.5, 171.5, 169.0, 166.6, 162.2 (d, J=244.8 Hz), 141.3, 139.2, 136.1,135.2, 129.4, 129.1, 129.0 (d, J=9.6 Hz), 128.4, 128.2, 126.5, 126.0,125.4, 115.9 (d, J=21.5 Hz), 50.4, 50.2, 39.1, 38.8, 38.7, 37.0, 31.1,28.5; ¹⁹F NMR (471 MHz, DMSO-d₆) δ −117.4 (m). HRMS calc. forC₃₂H₃₇FN₄O₄Na [M+Na]⁺: 583.2697. Found: 583.2701.

Example 13—Synthesis of PKS21028

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-(4-cyanophenyl)benzoic acid (6.7 mg, 30μmol) and PKS3072 (11.9 mg, 25 μmol). After completion of the reaction,the mixture was purified by HPLC to give product (11.0 mg, 78%) as awhite solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.61 (t, J=5.6 Hz, 1H), 8.19(t, J=1.9 Hz, 1H), 8.01 (d, J=8.0 Hz, 1H), 7.99-7.93 (m, 5H), 7.91 (d,J=7.8 Hz, 2H), 7.59 (t, J=7.8 Hz, 1H), 7.39 (s, 1H), 7.28-7.21 (m, 2H),7.19-7.13 (m, 3H), 4.54-4.46 (m, 1H), 3.45-3.15 (m, 4H), 2.76 (t, J=8.0Hz, 2H), 2.45 (dd, J=14.5, 5.9 Hz, 1H), 2.42-2.38 (m, 2H), 2.32 (dd,J=14.5, 7.9 Hz, 1H), 1.19 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ 171.3,171.2, 168.9, 166.1, 144.0, 141.3, 138.2, 135.4, 132.9, 129.7, 129.2,128.3, 128.1, 127.7, 127.7, 125.8, 125.7, 118.8, 110.4, 50.2, 50.0,38.9, 38.7, 38.6, 36.9, 30.9, 28.4.

Example 14—Synthesis of PKS21186

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 4-phenylpicolinic acid (10.0 mg, 50 μmol)and PKS3072 (23.8 mg, 50 μmol). After completion of the reaction, themixture was purified by HPLC to give product (21.5 mg, 79%) as a whitesolid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.96 (t, J=6.0 Hz, 1H), 8.69 (d,J=5.0 Hz, 1H), 8.28 (d, J=1.8 Hz, 1H), 7.97 (d, J=8.1 Hz, 1H), 7.95-7.90(m, 2H), 7.83 (d, J=6.8 Hz, 2H), 7.58-7.48 (m, 3H), 7.34 (s, 1H),7.27-7.21 (m, 2H), 7.19-7.13 (m, 3H), 4.54-4.44 (m, 1H), 3.43-3.38 (m,2H), 3.28-3.18 (m, 2H), 2.77 (t, J=7.9 Hz, 2H), 2.46-2.35 (m, 3H), 2.29(dd, J=14.6, 8.1 Hz, 1H), 1.20 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ171.2, 171.1, 168.8, 164.2, 150.8, 149.1, 148.6, 141.3, 136.7, 129.6,129.3, 128.3, 128.1, 126.9, 125.8, 123.7, 119.0, 50.1, 50.0, 38.9, 38.7,38.7, 36.9, 31.0, 28.4. HRMS calc. for C₃₁H₃₇N₅O₄Na [M+Na]⁺: 566.2743.Found: 566.2736.

Example 15—Synthesis of PKS21187

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-5-(2-fluorophenyl)benzoic acid(11.7 mg, 50 μmol) and PKS3072 (23.8 mg, 50 μmol). After completion ofthe reaction, the mixture was purified by HPLC to give product (24.8 mg,86%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.38 (t, J=4.6 Hz,1H), 7.96 (d, J=8.0 Hz, 1H), 7.92 (t, J=5.7 Hz, 1H), 7.82-7.78 (m, 1H),7.71-7.66 (m, 1H), 7.58-7.53 (m, 1H), 7.47-7.41 (m, 1H), 7.41-7.28 (m,4H), 7.27-7.22 (m, 2H), 7.19-7.13 (m, 3H), 4.53-4.42 (m, 1H), 3.38-3.29(m, 2H), 3.29-3.23 (m, 1H), 3.22-3.15 (m, 1H), 2.76 (t, J=7.9 Hz, 2H),2.46-2.36 (m, 3H), 2.30 (dd, J=14.6, 7.9 Hz, 1H), 1.18 (s, 9H); ¹³C NMR(126 MHz, DMSO-d₆) δ 171.3, 171.2, 168.8, 163.6, 159.0 (d, J=247.4 Hz),158.8 (d, J=251.0 Hz), 141.3, 132.8-132.6 (m), 131.4-131.1 (m), 130.8,130.3, 130.0 (d, J=7.9 Hz), 128.2, 128.1, 126.7, 125.8, 125.0 (d, J=2.9Hz), 124.2 (d, J=14.5 Hz), 116.5 (d, J=22.1 Hz), 116.1 (d, J=23.1 Hz),50.1, 50.0, 39.0, 38.6, 38.5, 36.9, 30.9, 28.4; ¹⁹F NMR (471 MHz,DMSO-d₆) δ −117.8 (m), −120.8 (m). HRMS calc. for C₃₂H₃₆F₂N₄O₄Na[M+Na]⁺: 601.2602. Found: 601.2601.

Example 16—Synthesis of PKS21195

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-5-(3-fluorophenyl)benzoic acid(14.3 mg, 61 μmol) and PKS3072 (29.0 mg, 61 μmol). After completion ofthe reaction, the mixture was purified by HPLC to give product (28.5 mg,81%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.41 (t, J=5.6 Hz,1H), 8.00 (d, J=8.0 Hz, 1H), 7.97-7.89 (m, 2H), 7.88-7.79 (m, 1H),7.59-7.53 (m, 2H), 7.53-7.46 (m, 1H), 7.41-7.32 (m, 2H), 7.27-7.18 (m,3H), 7.17-7.12 (m, 3H), 4.54-4.44 (m, 1H), 3.40-3.15 (m, 4H), 2.76 (t,J=8.0 Hz, 2H), 2.45 (dd, J=14.5, 6.0 Hz, 1H), 2.42-2.36 (m, 2H), 2.32(dd, J=14.5, 7.9 Hz, 1H), 1.18 (s, 9H); 13C NMR (126 MHz, DMSO-d₆) δ171.3, 171.2, 168.8, 163.7, 162.7 (d, J=243.3 Hz), 159.1 (d, J=250.7Hz), 141.3, 141.0 (d, J=7.7 Hz), 135.0, 130.9 (d, J=8.0 Hz), 130.5 (d,J=8.9 Hz), 128.3, 128.2, 128.1, 125.8, 124.5 (d, J=14.5 Hz), 122.8,116.8 (d, J=21.9 Hz), 114.4 (d, J=21.6 Hz), 113.4, 50.2, 50.0, 38.9,38.6, 38.6, 36.9, 30.9, 28.4; ¹⁹F NMR (471 MHz, DMSO-d₆) δ −114.7 (m),−117.9 (m). HRMS calc. for C₃₂H₃₆F₂N₄O₄Na [M+Na]⁺: 601.2602. Found:601.2600.

Example 17—Synthesis of PKS21196

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-5-phenyl-benzoic acid (13.2 mg,61 μmol) and PKS3072 (29.0 mg, 61 μmol). After completion of thereaction, the mixture was purified by HPLC to give product (30.0 mg,88%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.40 (t, J=5.6 Hz,1H), 8.00 (d, J=8.0 Hz, 1H), 7.94 (t, J=5.7 Hz, 1H), 7.90 (dd, J=6.9,2.4 Hz, 1H), 7.82-7.77 (m, 1H), 7.69 (d, J=7.6 Hz, 2H), 7.49-7.44 (m,2H), 7.40-7.33 (m, 3H), 7.27-7.21 (m, 2H), 7.18-7.13 (m, 3H), 4.54-4.45(m, 1H), 3.39-3.15 (m, 4H), 2.76 (t, J=8.0 Hz, 2H), 2.45 (dd, J=14.5,5.9 Hz, 1H), 2.42-2.37 (m, 2H), 2.32 (dd, J=14.5, 7.9 Hz, 1H), 1.18 (s,9H); ¹³C NMR (126 MHz, DMSO-d₆) δ 171.3, 171.2, 168.8, 163.8, 158.8 (d,J=250.7 Hz), 141.3, 138.5, 136.4, 130.3 (d, J=8.9 Hz), 129.0, 128.3,128.1 (d, J=3.1 Hz), 128.1, 127.7, 126.7, 125.8, 124.4 (d, J=14.5 Hz),116.7 (d, J=23.0 Hz), 50.2, 50.0, 38.9, 38.6, 38.6, 36.9, 30.9, 28.4;¹⁹F NMR (471 MHz, DMSO-d₆) δ −119.2 (m). HRMS calc. for C₃₂H₃₇FN₄O₄Na[M+Na]⁺: 583.2697. Found: 583.2697.

Example 18—Synthesis of PKS3086

The title compound was synthesized by following the general procedure ofHATU mediated coupling ofN-tert-butyl-N²-(1-oxo-3-phenylpropyl)-L-Aspargine (32.0 mg, 0.1 mmol)and tert-Butyl (2-aminopropyl)carbamate (17.4 mg, 0.1 mmol). Aftercompletion of the reaction, water was added. The white precipitateformed, was filtered and dried in air to give product (44.9 mg, 94%) asa white solid. Product was used in next step without furtherpurification. ¹H NMR (500 MHz, DMSO-d₆; A mixture of diastereomers) δ7.91 (d, J=8.0 Hz, 1H), 7.56 (d, J=8.1 Hz, 1H), 7.37 (s, 1H), 7.30-7.23(m, 2H), 7.22-7.13 (m, 3H), 6.70 (t, J=6.0 Hz, 1H), 4.50-4.41 (m, 1H),3.81-3.70 (m, 1H), 2.94 (t, J=6.1 Hz, 2H), 2.79 (t, J=7.9 Hz, 2H),2.45-2.34 (m, 3H), 2.30 (dd, J=14.7, 7.5 Hz, 1H), 1.37 (s, 9H), 1.22 (s,9H), 0.96 (d, J=6.7 Hz, 3H).

Example 19—Synthesis of PKS21006

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS3086 (40.0 mg, 84 μmol). Isolated crude wasdried under vacuum and triturated with diethyl ether to give a whitesolid. The diethylether was decanted and white solid was dried undervacuum to give product (40 mg, 97%) as a white solid. Product was usedin next step without further purification. ¹H NMR (500 MHz, DMSO-d₆; Amixture of diastereomers) δ 8.11 (d, J=7.2 Hz, 1H), 7.86 (d, J=8.4 Hz,1H), 7.55 (s, 1H), 7.30-7.24 (m, 2H), 7.22-7.15 (m, 3H), 4.46-4.32 (m,1H), 4.09-3.93 (m, 1H), 2.87 (dd, J=13.4, 5.1 Hz, 1H), 2.84-2.72 (m,3H), 2.47-2.39 (m, 4H), 1.23 (s, 9H), 1.11-1.03 (m, 3H).

Example 20—Synthesis of PKS21018

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-phenyl-benzoic acid (4.8 mg, 24 μmol)and PKS21006 (9.8 mg, 20 μmol). After completion of the reaction, themixture was purified by HPLC to give product (6.2 mg, 56%) as a whitesolid. ¹H NMR (500 MHz, DMSO-d₆; A mixture of diastereomers) δ 8.51 (t,J=5.9 Hz, 1H), 8.12 (t, J=1.9 Hz, 1H), 7.94 (d, J=7.8 Hz, 1H), 7.86-7.79(m, 2H), 7.77 (d, J=7.9 Hz, 1H), 7.74-7.69 (m, 2H), 7.53 (t, J=7.7 Hz,1H), 7.51-7.44 (m, 2H), 7.43-7.34 (m, 2H), 7.27-7.21 (m, 2H), 7.19-7.12(m, 3H), 4.52-4.44 (m, 1H), 4.00-3.91 (m, 1H), 3.44-3.36 (m, 1H),3.30-3.24 (m, 1H), 2.75 (t, J=8.0 Hz, 2H), 2.46-2.35 (m, 3H), 2.32 (dd,J=14.7, 7.6 Hz, 1H), 1.20 (s, 9H), 1.06 (d, J=6.6 Hz, 3H); ¹³C NMR (126MHz, DMSO-d₆) δ 171.2, 170.6, 168.8, 166.6, 141.2, 140.2, 139.6, 135.1,129.3, 129.0, 129.0, 128.9, 128.9, 128.2, 128.1, 127.7, 126.8, 126.4,125.8, 125.4, 50.3, 50.0, 45.1, 43.9, 38.6, 36.8, 30.9, 28.4, 17.7. HRMScalc. for C₃₃H₄₀N₄O₄Na [M+Na]⁺: 579.2947. Found: 579.2958.

Example 21—Synthesis of PKS3087

The title compound was synthesized by following the general procedure ofHATU mediated coupling ofN-tert-butyl-N²-(1-oxo-3-phenylpropyl)-L-Aspargine (32.0 mg, 0.1 mmol)and tert-Butyl N-(3-aminopropyl)carbamate (17.4 mg, 0.1 mmol). Aftercompletion of the reaction, water was added. The white precipitateformed, was filtered and dried in air to give product (44.3 mg, 93%) asa white solid. Product was used in next step without furtherpurification. ¹H NMR (500 MHz, DMSO-d₆) δ 7.99-7.94 (m, 1H), 7.69 (t,J=5.9 Hz, 1H), 7.34 (s, 1H), 7.29-7.23 (m, 2H), 7.21-7.14 (m, 3H), 6.74(t, J=5.9 Hz, 1H), 4.52-4.42 (m, 1H), 3.03-2.98 (m, 2H), 2.92-2.86 (m,2H), 2.82-2.77 (m, 2H), 2.46-2.36 (m, 3H), 2.28 (dd, J=14.6, 8.0 Hz,1H), 1.50-1.41 (m, 2H), 1.37 (s, 9H), 1.21 (s, 9H).

Example 22—Synthesis of PKS21007

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS3087 (44.3 mg, 93 μmol). After completion ofthe reaction (3 hours), excess trifluoroacetic acid and dichloromethanewere evaporated. Crude was dried under vacuum and triturated withdiethyl ether to give a white solid. The diethylether was decanted andthe white solid was dried under vacuum to give product (41.0 mg, 90%) asa white solid. Product was used in next step without furtherpurification. ¹H NMR (500 MHz, DMSO-d₆) δ 8.04 (d, J=7.8 Hz, 1H), 7.91(t, J=6.0 Hz, 1H), 7.73 (br, 3H), 7.41 (s, 1H), 7.33-7.22 (m, 2H),7.23-7.11 (m, 3H), 4.52-4.40 (m, 1H), 3.15-3.02 (m, 2H), 2.84-2.68 (m,4H), 2.46-2.37 (m, 3H), 2.32 (dd, J=14.7, 8.0 Hz, 1H), 1.72-1.59 (m,2H), 1.22 (s, 9H).

Example 23—Synthesis of PKS21019

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-phenyl-benzoic acid (4.8 mg, 24 μmol)and PKS21007 (9.8 mg, mol). After completion of the reaction, themixture was purified by HPLC to give product (11.1 mg, 72%) as a whitesolid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.57 (t, J=5.8 Hz, 1H), 8.11 (t,J=1.9 Hz, 1H), 8.00 (d, J=8.0 Hz, 1H), 7.86-7.78 (m, 3H), 7.75-7.69 (m,2H), 7.58-7.53 (m, 1H), 7.53-7.47 (m, 2H), 7.43-7.38 (m, 1H), 7.35 (s,1H), 7.27-7.21 (m, 2H), 7.21-7.12 (m, 3H), 4.50 (td, J=8.0, 5.8 Hz, 1H),3.31-3.26 (m, 2H), 3.15-3.08 (m, 2H), 2.84-2.77 (m, 2H), 2.47-2.38 (m,3H), 2.31 (dd, J=14.6, 8.0 Hz, 1H), 1.67-1.61 (m, 2H), 1.21 (s, 9H); ¹³CNMR (126 MHz, DMSO-d₆) δ 171.2, 171.0, 168.8, 166.1, 141.3, 140.2,139.6, 135.2, 129.3, 129.0, 129.0, 128.3, 128.1, 127.7, 126.8, 126.3,125.8, 125.3, 50.2, 50.0, 38.6, 36.9, 36.6, 36.3, 31.0, 29.1, 28.4. HRMScalc. for C₃₃H₄₀N₄O₄Na [M+Na]⁺: 579.2947. Found: 579.2953.

Example 24—Synthesis of PKS21017

The title compound was synthesized by following the general procedure ofHATU mediated coupling ofN-tert-butyl-N²-(1-oxo-3-phenylpropyl)-L-Glutamine (100.3 mg, 0.30 mmol)and N-boc-ethylenediamine (53.95 mg, 0.33 mmol). After completion of thereaction, water was added. The white precipitate formed, was filteredand dried in air to give product (105.0 mg, 73%) as a white solid.Product was used in the next step without further purification. ¹H NMR(500 MHz, DMSO-d₆) δ 7.95 (d, J=8.0 Hz, 1H), 7.85 (t, J=5.4 Hz, 1H),7.33 (s, 1H), 7.30-7.23 (m, 2H), 7.23-7.14 (m, 3H), 6.77 (t, J=5.0 Hz,1H), 4.18-4.09 (m, 1H), 3.13-3.00 (m, 2H), 3.00-2.93 (m, 2H), 2.80 (t,J=7.9 Hz, 2H), 2.48-2.40 (m, 2H), 1.97 (t, J=8.0 Hz, 2H), 1.80 (dt,J=13.9, 7.8, 7.2 Hz, 1H), 1.71-1.58 (m, 1H), 1.37 (s, 9H), 1.23 (s, 9H).

Example 25—Synthesis of PKS21021

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21017 (98.0 mg, 0.206 mmol). After completionof the reaction (3 hours), excess trifluoroacetic acid anddichloromethane were evaporated. Crude was dried under vacuum andtriturated with diethyl ether to give a white solid. The diethyletherwas decanted and white solid was dried under vacuum to give product(100.0 mg, 99%) as a white solid. Product was used in next step withoutfurther purification. ¹H NMR (500 MHz, DMSO-d₆) δ 8.09-8.01 (m, 2H),7.76 (s, 3H), 7.37 (s, 1H), 7.30-7.24 (m, 2H), 7.22-7.14 (m, 3H),4.16-4.08 (m, 1H), 3.32-3.25 (m, 2H), 2.89-2.77 (m, 4H), 2.48-2.38 (m,2H), 2.01 (t, J=7.9 Hz, 2H), 1.90-1.79 (m, 1H), 1.73-1.62 (m, 1H), 1.23(s, 9H).

Example 26—Synthesis of PKS21030

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-phenyl-benzoic acid (7.1 mg, 36 μmol)and PKS21021 (11.3 mg, 30.0 μmol). After completion of the reaction, themixture was purified by HPLC to give product (10.0 mg, 60%) as a whitesolid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.61 (t, J=5.5 Hz, 1H), 8.13 (t,J=1.8 Hz, 1H), 8.05-7.98 (m, 2H), 7.86-7.79 (m, 2H), 7.76-7.70 (m, 2H),7.54 (t, J=7.7 Hz, 1H), 7.52-7.45 (m, 2H), 7.42-7.37 (m, 1H), 7.34 (s,1H), 7.29-7.22 (m, 2H), 7.20-7.12 (m, 3H), 4.16 (td, J=8.2, 5.6 Hz, 1H),3.44-3.19 (m, 4H), 2.78 (t, J=7.9 Hz, 2H), 2.47-2.40 (m, 2H), 1.99 (t,J=8.0 Hz, 2H), 1.89-1.78 (m, 1H), 1.74-1.63 (m, 1H), 1.21 (s, 9H); ¹³CNMR (126 MHz, DMSO-d₆) δ 171.7, 171.4, 171.1, 166.4, 141.3, 140.2,139.6, 135.1, 129.3, 129.0, 129.0, 128.2, 128.1, 127.8, 126.8, 126.4,125.8, 125.4, 52.5, 49.8, 39.1, 38.5, 36.8, 32.5, 31.0, 28.5, 28.1.

Example 27—Synthesis of PKS21176

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 5-methylisoxazole-3-carboxylic acid (139.8mg, 1.10 mmol) and N-tert-butyl-L-Aspargine benzyl ester (TFA salt;431.0 mg, 1.10 mmol). After completion of the reaction, water was added.The white precipitate formed, was filtered, washed with water and driedin air to give product (395 mg, 93%) as a white solid. Product was pure(by NMR) and used in next step without further purification. ¹H NMR (500MHz, Chloroform-d) δ 8.00 (d, J=8.3 Hz, 1H), 7.37-7.26 (m, 5H), 6.40 (d,J=1.1 Hz, 1H), 5.30 (br, 1H), 5.25 (d, J=12.4 Hz, 1H), 5.19 (d, J=12.4Hz, 1H), 4.97 (dt, J=8.7, 4.5 Hz, 1H), 2.89 (dd, J=15.6, 4.6 Hz, 1H),2.71 (dd, J=15.6, 4.5 Hz, 1H), 2.47 (s, 3H), 1.28 (s, 9H).

Example 28—Synthesis of PKS21178

The title compound was synthesized by following the general procedurefor 0-debenzylation of PKS21176 (195.0 mg, 0.503 mmol). After completionof the reaction, the mixture was filtered through celite. Filtrate wasevaporated and dried to give product (146 mg, 98%) as a colorless gum.Product was used in next step without further purification. ¹H NMR (500MHz, Chloroform-d) δ 8.04 (d, J=6.5 Hz, 1H), 6.46 (s, 1H), 6.37 (s, 1H),4.83-4.75 (m, 1H), 2.93 (dd, J=15.6, 3.6 Hz, 1H), 2.78 (dd, J=15.6, 8.1Hz, 1H), 2.45 (s, 3H), 1.32 (s, 9H).

Example 29—Synthesis of PKS21184

The title compound was synthesized by following the general procedure ofHATU mediated coupling of PKS21178 (145.0 mg, 0.488 mmol) and tert-butylN-(2-aminoethyl)carbamate (78.1 mg, 0.488 mmol). After completion of thereaction, water was added. Mixture was extracted with ethyl acetatetwice. Combined organic layer was washed with aq. NaHCO₃, water, 1N HCl,saturated brine, dried over anhydrous sodium sulfate and evaporated togive product (210.0 mg, 98%) as an off-white solid. Product was used innext step without further purification. ¹H NMR (500 MHz, Chloroform-d) δ8.32 (d, J=7.7 Hz, 1H), 7.44 (br, 1H), 6.41 (s, 1H), 6.05-5.82 (m, 1H),5.13 (br, 1H), 4.90-4.80 (m, 1H), 3.41-3.29 (m, 2H), 3.28-3.18 (m, 2H),2.86-2.76 (m, 1H), 2.65-2.56 (m, 1H), 2.46 (s, 3H), 1.39 (s, 9H), 1.32(s, 9H).

Example 30—Synthesis of PKS21185

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21184 (210.0 mg, 0.478 mmol). Isolated crudewas dried under vacuum and triturated with diethyl ether to give a whitesolid. The diethylether was decanted and white solid was dried undervacuum to give product (197.0 mg, 91%) as a white solid. Product wasused in the next step without further purification. ¹H NMR (500 MHz,DMSO-d6) δ 8.54 (d, J=8.1 Hz, 1H), 8.25-8.16 (m, 1H), 7.78 (br, 3H),7.60 (s, 1H), 6.56 (d, J=1.0 Hz, 1H), 4.73-4.62 (m, 1H), 3.35-3.23 (m,2H), 2.89-2.81 (m, 2H), 2.59 (dd, J=13.3, 5.9 Hz, 1H), 2.55 (dd, J=13.3,4.8 Hz, 1H), 2.47 (s, 3H), 1.20 (s, 9H).

Example 31—Synthesis of PKS21208

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-5-(2-fluorophenyl)benzoic acid(11.7 mg, 50 μmol) and PKS21185 (22.7 mg, 50 μmol). After completion ofthe reaction, the mixture was purified by preparative LCMS to giveproduct (21.0 mg, 76%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ8.52 (d, J=8.0 Hz, 1H), 8.38 (d, J=5.9 Hz, 1H), 8.16 (t, J=5.6 Hz, 1H),7.78 (d, J=6.7 Hz, 1H), 7.72-7.67 (m, 1H), 7.60-7.54 (m, 1H), 7.49 (s,1H), 7.47-7.42 (m, 1H), 7.42-7.36 (m, 1H), 7.36-7.28 (m, 2H), 6.49 (s,1H), 4.69-4.61 (m, 1H), 3.43-3.15 (m, 4H), 2.56 (dd, J=14.4, 8.2 Hz,1H), 2.52-2.46 (m, 1H), 2.44 (s, 3H), 1.17 (s, 9H); ¹³C NMR (126 MHz,DMSO-d₆) δ 171.2, 170.4, 168.9, 163.5, 159.0 (d, J=247.1 Hz), 158.8 (d,J=251.1 Hz), 158.6, 158.3, 132.8-132.5 (m), 131.3, 130.8 (d, J=3.3 Hz),130.4, 130.0 (d, J=7.4 Hz), 126.6 (d, J=12.7 Hz), 125.0 (d, J=2.7 Hz),124.1 (d, J=14.6 Hz), 116.5 (d, J=23.0 Hz), 116.1 (d, J=21.8 Hz), 101.3,50.5, 50.1, 39.0, 38.5, 38.1, 28.3, 11.8; ¹⁹F NMR (471 MHz, DMSO-d₆)−117.8 (m), −120.8 (m). HRMS calc. for C₂₈H₃₁F₂N₅O₅Na [M+Na]⁺: 578.2191.Found: 578.2177.

Example 32—Synthesis of PKS21224

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 4-phenylpyridine-2-carboxylic acid (10.0mg, 50 μmol) and PKS21185 (22.7 mg, 50 μmol). After completion of thereaction, the mixture was purified by preparative LCMS to give product(18.2 mg, 70%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.95 (t,J=6.0 Hz, 1H), 8.68 (d, J=5.0 Hz, 1H), 8.53 (d, J=8.0 Hz, 1H), 8.25 (s,1H), 8.18 (t, J=5.6 Hz, 1H), 7.94-7.90 (m, 1H), 7.84 (d, J=7.3 Hz, 2H),7.59-7.46 (m, 4H), 6.51 (s, 1H), 4.70-4.62 (m, 1H), 3.49-3.19 (m, 4H),2.55 (dd, J=14.5, 8.3 Hz, 1H), 2.50-2.45 (m, 1H), 2.43 (s, 3H), 1.17 (s,9H); ¹³C NMR (126 MHz, DMSO-d₆) δ 171.1, 170.4, 168.9, 164.2, 158.6,158.2, 150.7, 149.1, 148.6, 136.7, 129.6, 129.3, 126.9, 123.7, 119.0,101.3, 50.4, 50.1, 38.9, 38.7, 38.1, 28.3, 11.8. HRMS calc. forC₂₇H₃₂N₆O₅Na [M+Na]⁺: 543.2332. Found: 543.2315.

Example 33—Synthesis of PKS21225

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-5-(3-fluorophenyl)benzoic acid(11.7 mg, 50 μmol) and PKS21185 (22.7 mg, 50 μmol). After completion ofthe reaction, the mixture was purified by preparative LCMS to giveproduct (24.4 mg, 88%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ8.53 (d, J=8.0 Hz, 1H), 8.41 (t, J=5.6 Hz, 1H), 8.18 (t, J=5.6 Hz, 1H),7.93-7.89 (m, 1H), 7.87-7.82 (m, 1H), 7.59-7.47 (m, 4H), 7.42-7.31 (m,1H), 7.24-7.18 (m, 1H), 6.49 (s, 1H), 4.71-4.62 (m, 1H), 3.42-3.17 (m,4H), 2.57 (dd, J=14.4, 8.2 Hz, 1H), 2.53-2.46 (m, 1H), 2.44 (s, 3H),1.17 (s, 9H). HRMS calc. for C₂₈H₃₁F₂N₅O₅Na [M+Na]⁺: 578.2191. Found:578.2183.

Example 34—Synthesis of PKS21250

The title compound was synthesized by following the general procedurefor HATU mediated coupling of picolinic acid (6.2 mg, 50 μmol) andPKS21185 (22.7 mg, 50 mol). After completion of the reaction, themixture was purified by preparative LCMS to give product (20.4 mg, 92%)as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.88 (t, J=6.0 Hz, 1H),8.62 (d, J=4.7 Hz, 1H), 8.52 (d, J=8.0 Hz, 1H), 8.21-8.11 (m, 1H),8.04-7.93 (m, 2H), 7.59 (dd, J=7.0, 4.6 Hz, 1H), 7.49 (s, 1H), 6.52 (s,1H), 4.70-4.59 (m, 1H), 3.43-3.29 (m, 2H), 3.29-3.16 (m, 2H), 2.54 (dd,J=14.3, 8.3 Hz, 1H), 2.49-2.43 (m, 4H), 1.17 (s, 9H); ¹³C NMR (126 MHz,DMSO-d₆) δ 171.2, 170.4, 168.9, 164.2, 158.6, 158.2, 149.9, 148.3,137.7, 126.4, 121.89, 101.3, 50.4, 50.1, 38.9, 38.6, 38.1, 28.3, 11.8.HRMS calc. for C₂₁H₂₈N₆O₅Na [M+Na]⁺: 467.2019. Found: 467.2003.

Example 35—Synthesis of PKS21251

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluorobenzoic acid (7.0 mg, 50 μmol) andPKS21185 (22.7 mg, 50 μmol). After completion of the reaction, themixture was purified by preparative LCMS to give product (19.8 mg, 86%)as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.53 (d, J=7.9 Hz, 1H),8.26 (t, J=5.8 Hz, 1H), 8.14 (t, J=5.7 Hz, 1H), 7.66-7.59 (m, 1H),7.56-7.46 (m, 2H), 7.30-7.21 (m, 2H), 6.52 (s, 1H), 4.70-4.60 (m, 1H),3.39-3.14 (m, 4H), 2.56 (dd, J=14.5, 8.1 Hz, 1H), 2.49-2.44 (m, 4H),1.18 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ 171.2, 170.4, 168.9, 163.8,159.1 (d, J=250.8 Hz), 158.6, 158.3, 132.4 (d, J=7.8 Hz), 130.2, 124.4,123.9 (d, J=14.4 Hz), 116.0 (d, J=23.4 Hz), 101.3, 50.4, 50.1, 38.9,38.5, 38.1, 28.3, 11.8; ¹⁹F NMR (471 MHz, DMSO-d₆) δ −116.5 (m). HRMScalc. for C₂₂H₂₈FN₅O₅Na [M+Na]⁺: 484.1972. Found: 484.1969.

Example 36—Synthesis of PKS21212

N-tert-butyl-N²-Boc-L-Aspargine benzyl ester (1.60 g, 4.23 mmol) wasdissolved in water: tetrahydrofuran (1:1, 20 mL) mixture and 5 mL of HCl(12 N) was added. The mixture was stirred at room temperature for 4hours. Tetrahydrofuran was evaporated and the resulting solution wasdiluted with 10 mL water and basified with pinch-wise addition of solidsodium bicarbonate (approx. 12 g). 4-Toluenesulfonyl chloride (1.61 g,8.46 mmol) and 50 mL ethyl acetate were added. The biphasic mixture wasvigorously stirred at room temperature for 2 hours. The layers wereseparated and aqueous layer was washed with ethyl acetate. Combinedethyl acetate layer was evaporated and purified by combi-flash to giveproduct (1.37 g, 75%) as a white solid. ¹H NMR (500 MHz, Chloroform-d) δ7.75-7.68 (m, 2H), 7.35-7.28 (m, 3H), 7.22 (d, J=7.9 Hz, 5H), 5.90 (d,J=7.8 Hz, 1H), 5.31 (s, 1H), 5.04 (d, J=12.2 Hz, 1H), 5.00 (d, J=12.2Hz, 1H), 4.10 (dt, J=8.3, 4.3 Hz, 1H), 2.80 (dd, J=15.3, 4.1 Hz, 1H),2.62 (dd, J=15.3, 4.6 Hz, 1H), 2.39 (s, 3H), 1.29 (s, 9H).

Example 37—Synthesis of PKS21241

The title compound was synthesized by following the general procedurefor 0-debenzylation of PKS21212 (1.37 g, 3.17 mmol) in tetrahydrofuran(15.00 mL). After completion of the reaction, the mixture was filteredthrough celite. Filtrate was evaporated and dried to give product (1.06g, 98%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 12.55 (s, 1H),7.86 (d, J=8.7 Hz, 1H), 7.66 (d, J=7.8 Hz, 2H), 7.41 (s, 1H), 7.33 (d,J=7.8 Hz, 2H), 4.09-4.02 (m, 1H), 2.42 (dd, J=15.1, 6.8 Hz, 1H), 2.36(s, 3H), 2.24 (dd, J=15.1, 6.5 Hz, 1H), 1.17 (s, 9H).

Example 38—Synthesis of PKS21177

The title compound was synthesized by following the general procedure ofHATU mediated coupling of Ts-Asp(CONHtBu)-OH (342.4 mg, 1.00 mmol) andN-Boc-ethylenediamine (176.2 mg, 1.10 mmol). After completion of thereaction, water was added and mixture was stirred for 30 minutes at roomtemperature. The white precipitate formed, was filtered, washed withwater and dried in air to give product (441.0 mg, 91%) as a white solid.Product was used in next step without further purification. ¹H NMR (500MHz, Chloroform-d) δ 7.76 (d, J=8.4 Hz, 2H), 7.34-7.30 (m, 3H), 6.71(br, 1H), 5.51 (br, 1H), 4.98 (br, 1H), 3.93-3.84 (m, 1H), 3.34-3.26 (m,2H), 3.23-3.15 (m, 2H), 2.67 (dd, J=15.1, 4.2 Hz, 1H), 2.43 (s, 3H),2.16-2.04 (m, 1H), 1.45 (s, 9H), 1.27 (s, 9H).

Example 39—Synthesis of PKS21183

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21177 (431.0 mg, 0.889 mmol). Aftercompletion of the reaction (3 hours), excess trifluoroacetic acid anddichloromethane were evaporated. Crude was dried under vacuum andtriturated with diethyl ether to give a white solid. The diethyletherwas decanted and a white solid was dried under vacuum to give product(440.0 mg, 99%) as a white solid. Product was used in next step withoutfurther purification. ¹H NMR (500 MHz, DMSO-d₆) δ 8.07 (t, J=5.9 Hz,1H), 7.88 (d, J=8.4 Hz, 1H), 7.69 (s, 3H), 7.66 (d, J=8.0 Hz, 2H), 7.57(s, 1H), 7.34 (d, J=8.0 Hz, 2H), 3.96-3.87 (m, 1H), 3.21-3.11 (m, 1H),3.10-3.00 (m, 1H), 2.80-2.64 (m, 2H), 2.37 (s, 3H), 2.34 (dd, J=14.8,7.9 Hz, 1H), 2.27 (dd, J=14.8, 6.1 Hz, 1H), 1.18 (s, 9H).

Example 40—Synthesis of PKS21221

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-5-(2-fluorophenyl)benzoic acid(11.7 mg, 50 μmol) and PKS21183 (24.9 mg, 50 μmol). After completion ofthe reaction, the mixture was purified by preparative LCMS to giveproduct (25.0 mg, 83%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ8.27 (t, J=5.7 Hz, 1H), 7.95 (t, J=5.8 Hz, 1H), 7.80-7.74 (m, 2H),7.72-7.67 (m, 1H), 7.64 (d, J=7.5 Hz, 2H), 7.59-7.53 (m, 1H), 7.48-7.28(m, 7H), 4.02-3.91 (m, 1H), 3.24-3.11 (m, 2H), 3.09-2.93 (m, 2H), 2.35(s, 3H), 2.31 (dd, J=14.7, 7.0 Hz, 1H), 2.20 (dd, J=14.7, 6.9 Hz, 1H),1.14 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ 170.1, 168.1, 163.4, 159.0(d, J=247.2 Hz), 158.8 (d, J=251.2 Hz), 142.5, 138.1, 132.8-132.6 (m),131.3 (d, J=2.5 Hz), 130.8, 130.3, 130.0 (d, J=7.5 Hz), 129.2, 126.6,126.6, 125.0 (d, J=2.8 Hz), 124.1 (d, J=14.7 Hz), 116.5 (d, J=23.5 Hz),116.2 (d, J=23.1 Hz), 53.7, 50.1, 39.4, 38.8, 38.2, 28.3, 20.9; ¹⁹F NMR(471 MHz, DMSO-d₆) δ −117.8 (m), −120.9 (m). HRMS calc. forC₃OH₃₄F₂N₄O₅SNa [M+Na]⁺: 623.2116. Found: 623.2107.

Example 41—Synthesis of PKS21228

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-5-(3-fluorophenyl)benzoic acid(11.7 mg, 50 μmol) and PKS21183 (24.9 mg, 50 μmol). After completion ofthe reaction, the mixture was purified by preparative LCMS to giveproduct (24.5 mg, 82%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ8.31 (t, J=5.7 Hz, 1H), 7.98 (t, J=5.9 Hz, 1H), 7.91 (dd, J=6.7, 2.3 Hz,1H), 7.87-7.82 (m, 1H), 7.78 (br, 1H), 7.65 (d, J=7.4 Hz, 2H), 7.59-7.46(m, 3H), 7.41-7.35 (m, 2H), 7.31 (d, J=7.9 Hz, 2H), 7.25-7.18 (m, 1H),4.02-3.92 (m, 1H), 3.26-3.12 (m, 2H), 3.11-2.93 (m, 2H), 2.35 (s, 3H),2.34-2.29 (m, 1H), 2.20 (dd, J=14.7, 6.8 Hz, 1H), 1.14 (s, 9H); ¹³C NMR(126 MHz, DMSO-d₆) δ 170.2, 168.1, 163.5, 162.7 (d, J=243.9 Hz), 159.1(d, J=252.7 Hz), 142.5, 140.9 (d, J=7.3 Hz), 138.1, 135.0, 130.9 (d,J=8.9 Hz), 130.6 (d, J=8.9 Hz), 129.2, 128.3, 126.6, 124.5 (d, J=14.5Hz), 122.8, 116.8 (d, J=21.8 Hz), 114.4 (d, J=20.2 Hz), 113.5 (d, J=21.9Hz), 53.7, 50.1, 39.3, 38.7, 38.3, 28.3, 20.9; ¹⁹F NMR (471 MHz,DMSO-d₆) δ −114.9 (m), −118.2 (m). HRMS calc. for C₃₀H₃₄F₂N₄O₅SNa[M+Na]⁺: 623.2116. Found: 623.2117.

Example 42—Synthesis of PKS21229

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 4-phenylpyridine-2-carboxylic acid (10.0mg, 50 μmol) and PKS21183 (24.9 mg, 50 μmol). After completion of thereaction, the mixture was purified by preparative LCMS to give product(16.0 mg, 57%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.91 (t,J=6.0 Hz, 1H), 8.73 (d, J=5.0 Hz, 1H), 8.31 (s, 1H), 8.03 (t, J=5.7 Hz,1H), 7.96 (dd, J=4.9, 2.3 Hz, 1H), 7.88 (d, J=7.4 Hz, 2H), 7.79 (br,1H), 7.67 (d, J=7.8 Hz, 2H), 7.62-7.50 (m, 3H), 7.38 (s, 1H), 7.30 (d,J=7.8 Hz, 2H), 4.06-3.95 (m, 1H), 3.34-3.22 (m, 2H), 3.14-2.99 (m, 2H),2.35 (s, 3H), 2.34-2.30 (m, 1H), 2.22 (dd, J=14.6, 7.1 Hz, 1H), 1.18 (s,9H); ¹³C NMR (126 MHz, DMSO-d₆) δ 170.1, 168.0, 164.0, 150.8, 149.1,148.6, 142.4, 138.1, 136.7, 129.7, 129.4, 129.2, 126.9, 126.6, 123.7,119.0, 53.7, 50.1, 39.5, 38.6, 38.4, 28.4, 20.9. HRMS calc. forC₂₉H₃₅N₅O₅SNa [M+Na]⁺: 588.2257. Found: 588.2238.

Example 43—Synthesis of PKS21282

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-(1H-pyrazol-3-yl)benzoic acid (6.2 mg,33 μmol) and PKS21183 (15.0 mg, 30 μmol). After completion of thereaction, the mixture was purified by preparative LCMS to give product(10.6 mg, 64%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.40 (t,J=5.6 Hz, 1H), 8.27-8.22 (m, 1H), 8.00 (t, J=5.7 Hz, 1H), 7.95 (dt,J=7.7, 1.4 Hz, 1H), 7.79 (d, J=8.8 Hz, 1H), 7.77-7.75 (m, 1H), 7.74 (dt,J=7.7, 1.4 Hz, 1H), 7.65 (d, J=8.0 Hz, 2H), 7.49 (t, J=7.7 Hz, 1H), 7.40(s, 1H), 7.29 (d, J=8.0 Hz, 2H), 6.76 (d, J=2.2 Hz, 1H), 4.04-3.96 (m,1H), 3.23-3.15 (m, 2H), 3.11-2.95 (m, 2H), 2.34 (s, 3H), 2.33 (dd,J=14.6. 7.23 Hz, 1H), 2.21 (dd, J=14.6, 6.8 Hz, 1H), 1.16 (s, 9H); ¹³CNMR (126 MHz, DMSO-d₆) δ 170.2, 168.1, 166.2, 148.0, 142.5, 138.1,134.9, 133.0, 131.9, 129.3, 128.7, 127.7, 126.6, 126.1, 124.0, 102.2,53.7, 50.1, 39.5, 38.8, 38.4, 28.4, 21.0.

Example 44—Synthesis of PKS21291

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-benzoic acid (4.6 mg, 33 μmol)and PKS21183 (15.0 mg, mol). After completion of the reaction, themixture was purified by preparative LCMS to give product (13.9 mg, 91%)as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.22-8.13 (m, 1H), 7.95(t, J=5.7 Hz, 1H), 7.78 (br, 1H), 7.68-7.60 (m, 3H), 7.57-7.47 (m, 1H),7.38 (s, 1H), 7.35-7.23 (m, 4H), 4.03-3.92 (m, 1H), 3.24-3.08 (m, 2H),3.08-2.92 (m, 2H), 2.35 (s, 3H), 2.31 (dd, J=14.6, 7.1 Hz, 1H), 2.19(dd, J=14.6, 6.8 Hz, 1H), 1.15 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ170.1, 168.1, 163.7, 159.1 (d, J=249.0 Hz), 142.5, 138.1, 132.4 (d,J=8.8 Hz), 130.2, 129.2, 126.6, 124.4 (d, J=2.5 Hz), 123.9 (d, J=14.5Hz), 116.1 (d, J=21.8 Hz), 53.7, 50.1, 39.4, 38.7, 38.2, 28.4, 20.9; ¹⁹FNMR (471 MHz, DMSO-d₆) δ −116.5 (m).

Example 45—Synthesis of PKS21295

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-(2-oxoimidazolidin-1-yl)benzoic acid(6.80 mg, 33 μmol) and PKS21183 (15.0 mg, 30 μmol). After completion ofthe reaction, the mixture was purified by preparative LCMS to giveproduct (14.1 mg, 82%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ8.29 (t, J=5.6 Hz, 1H), 7.99 (t, J=5.7 Hz, 1H), 7.86-7.80 (m, 2H), 7.79(br, 1H), 7.64 (d, J=8.2 Hz, 2H), 7.45-7.39 (m, 2H), 7.37 (t, J=7.8 Hz,1H), 7.29 (d, J=8.2 Hz, 2H), 7.03 (s, 1H), 3.99 (t, J=6.9 Hz, 1H),3.91-3.84 (m, 2H), 3.45-3.39 (m, 2H), 3.20-3.12 (m, 2H), 3.08-2.92 (m,2H), 2.38-2.28 (m, 1H), 2.33 (s, 3H), 2.20 (dd, J=14.5, 6.9 Hz, 1H),1.16 (s, 9H). ¹³C NMR (126 MHz, DMSO-d₆) δ 170.2, 168.1, 166.4, 158.9,142.5, 140.8, 138.1, 135.0, 129.2, 128.4, 126.6, 120.0, 119.7, 115.6,53.7, 50.1, 44.5, 39.5, 38.8, 38.4, 36.5, 28.4, 20.9.

Example 46—Synthesis of PKS21315

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 1H-indole-2-carboxylic acid (4.83 mg,30.00 μmol) and PKS21183 (15.0 mg, 30 μmol). After completion of thereaction, the mixture was purified by preparative LCMS to give product(9.9 mg, 63%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.58 (d,J=2.2 Hz, 1H), 8.36 (t, J=5.7 Hz, 1H), 8.02 (t, J=5.8 Hz, 1H), 7.81 (br,1H), 7.65 (d, J=8.0 Hz, 2H), 7.59 (d, J=7.9 Hz, 1H), 7.45-7.39 (m, 2H),7.29 (d, J=8.0 Hz, 2H), 7.17 (t, J=7.6 Hz, 1H), 7.08 (d, J=1.9 Hz, 1H),7.03 (t, J=7.4 Hz, 1H), 4.04-3.96 (m, 1H), 3.22-3.15 (m, 2H), 3.09-2.93(m, 2H), 2.38-2.29 (m, 4H), 2.22 (dd, J=14.5, 6.8 Hz, 1H), 1.18 (s, 9H).¹³C NMR (126 MHz, DMSO-d₆) δ 170.2, 168.1, 161.1, 142.5, 138.1, 136.4,131.7, 129.2, 127.1, 126.6, 123.2, 121.4, 119.7, 112.3, 102.4, 53.8,50.1, 39.5, 38.5, 38.2, 28.4, 21.0.

Example 47—Synthesis of PKS21242

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-(4-fluorophenyl)propanoic acid (223.7mg, 1.33 mmol) and N-tert-butyl-L-Aspargine benzyl ester (TFA salt;521.9 mg, 1.33 mmol). After completion of the reaction, water was added.The white precipitate formed, was filtered, washed with water and driedin air to give product (515.0 mg, 90%) as a white solid. Product waspure (by NMR) and used in next step without further purification. ¹H NMR(500 MHz, Chloroform-d) δ 7.37-7.28 (m, 5H), 7.18-7.09 (m, 2H),6.98-6.90 (m, 2H), 6.88 (d, J=8.1 Hz, 1H), 5.28 (s, 1H), 5.20 (d, J=12.3Hz, 1H), 5.14 (d, J=12.3 Hz, 1H), 4.84-4.76 (m, 1H), 2.92 (t, J=7.9 Hz,2H), 2.80 (dd, J=15.8, 3.8 Hz, 1H), 2.56-2.44 (m, 3H), 1.27 (s, 9H).

Example 48—Synthesis of PKS21243

The title compound was synthesized by following the general procedurefor 0-debenzylation of PKS21242 (515.0 mg, 1.20 mmol). After completionof the reaction, the mixture was filtered through celite. Filtrate wasevaporated and dried to give product (405.0 mg, quant.) as a whitesolid. Product was used in next step without further purification. ¹HNMR (500 MHz, DMSO-d₆) δ 12.50 (s, 1H), 8.04 (d, J=7.8 Hz, 1H), 7.42 (s,1H), 7.27-7.16 (m, 2H), 7.10-7.01 (m, 2H), 4.52-4.41 (m, 1H), 2.78 (t,J=7.7 Hz, 2H), 2.49-2.44 (m, 1H), 2.42-2.34 (m, 3H), 1.22 (s, 9H).

Example 49—Synthesis of PKS21246

The title compound was synthesized by following the general procedure ofHATU mediated coupling of PKS21243 (310.0 mg, 0.916 mmol) andN-Boc-ethylenediamine (146.8 mg, 0.916 mmol). After completion of thereaction, water was added. The white precipitate formed, was filtered,washed with water and dried in air to give product (393.0 mg, 89%) as awhite solid. Product was used in next step without further purification.¹H NMR (500 MHz, DMSO-d₆) δ 7.94 (d, J=8.0 Hz, 1H), 7.80 (t, J=5.9 Hz,1H), 7.35 (s, 1H), 7.27-7.18 (m, 2H), 7.11-7.02 (m, 2H), 6.75 (t, J=5.7Hz, 1H), 4.51-4.39 (m, 1H), 3.13-2.90 (m, 4H), 2.78 (t, J=7.8 Hz, 2H),2.43-2.36 (m, 3H), 2.29 (dd, J=14.7, 7.8 Hz, 1H), 1.37 (s, 9H), 1.21 (s,9H).

Example 50—Synthesis of PKS21249

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21246 (390.0 mg, 0.812 mmol). Isolated crudewas dried under vacuum and triturated with diethyl ether. Diethyl etherwas decanted and white solid was dried to give product (400 mg, quant.).Product was used in next step without further purification. ¹H NMR (500MHz, DMSO-d₆) δ 8.08-8.03 (m, 2H), 7.76 (br, 3H), 7.56 (s, 1H),7.26-7.17 (m, 2H), 7.10-7.02 (m, 2H), 4.50-4.38 (m, 1H), 3.40-3.30 (m,1H), 3.28-3.16 (m, 1H), 2.92-2.81 (m, 2H), 2.78 (t, J=7.9 Hz, 2H),2.47-2.31 (m, 4H), 1.22 (s, 9H).

Example 51—Synthesis of PKS21254

The title compound was synthesized by following the general procedurefor HATU mediated coupling of picolinic acid (6.2 mg, 51 μmol) andPKS21249 (25.0 mg, 51 mol). After completion of the reaction, themixture was purified by preparative LCMS to give product (19.5 mg, 79%)as an off-white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.90 (t, J=6.1 Hz,1H), 8.62 (d, J=4.5 Hz, 1H), 8.02 (d, J=7.8 Hz, 1H), 8.00-7.90 (m, 3H),7.63-7.54 (m, 1H), 7.34 (s, 1H), 7.26-7.16 (m, 2H), 7.13-7.01 (m, 2H),4.54-4.43 (m, 1H), 3.41-3.34 (m, 2H), 3.29-3.15 (m, 2H), 2.76 (t, J=7.9Hz, 2H), 2.45-2.32 (m, 3H), 2.28 (dd, J=14.5, 8.0 Hz, 1H), 1.19 (s, 9H);¹³C NMR (126 MHz, DMSO-d₆) δ 171.2, 171.0, 168.8, 164.2, 160.6 (d,J=240.0 Hz), 149.9, 148.3, 137.7, 137.4 (d, J=2.7 Hz), 129.9 (d, J=8.7Hz), 126.5, 121.9, 114.9 (d, J=21.0 Hz), 50.1, 50.0, 38.8, 38.7, 38.6,36.9, 30.1, 28.4. HRMS calc. for C₂₅H₃₂FN₅O₄Na [M+Na]⁺: 508.2336. Found:508.2324.

Example 52—Synthesis of PKS21255

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluorobenzoic acid (7.1 mg, 51 μmol) andPKS21249 (25.0 mg, 51 μmol). After completion of the reaction, themixture was purified by preparative LCMS to give product (20.8 mg, 82%)as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.27 (t, J=5.8 Hz, 1H),7.97 (d, J=8.1 Hz, 1H), 7.95-7.90 (m, 1H), 7.65 (t, J=7.6 Hz, 1H),7.56-7.46 (m, 1H), 7.36 (s, 1H), 7.30-7.23 (m, 2H), 7.23-7.17 (m, 2H),7.10-7.03 (m, 2H), 4.52-4.41 (m, 1H), 3.32-3.28 (m, 2H), 3.27-3.13 (m,2H), 2.76 (t, J=7.9 Hz, 2H), 2.46-2.35 (m, 3H), 2.29 (dd, J=14.8, 7.8Hz, 1H), 1.22-1.13 (m, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ 171.3, 171.1,168.8, 163.9, 160.6 (d, J=240.3 Hz), 159.1 (d, J=249.3 Hz), 137.4, 132.4(d, J=9.0 Hz), 130.2 (d, J=1.6 Hz), 129.9, 124.4 (d, J=2.7 Hz), 123.9(d, J=14.4 Hz), 116.1 (d, J 23.4 Hz), 114.9 (d, J=21.4 Hz), 50.1, 50.0,38.9, 38.6, 38.5, 36.9, 30.1, 28.4; ¹⁹F NMR (471 MHz, DMSO-d₆) δ −116.5(m), −119.8 (m). HRMS calc. for C₂₆H₃₂F₂N₄O₄Na [M+Na]⁺: 521.2289. Found:521.2311.

Example 53—Synthesis of PKS21258

The title compound was synthesized by following the general procedurefor HATU mediated coupling of quinoline-8-carboxylic acid (8.8 mg, 51μmol) and PKS21249 (25.0 mg, 51 μmol). After completion of the reaction,the mixture was purified by preparative LCMS to give product (18.8 mg,69%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 10.91 (t, J=5.7 Hz,1H), 9.07 (dd, J=4.3, 2.1 Hz, 1H), 8.59-8.50 (m, 2H), 8.19 (dd, J=8.1,2.1 Hz, 1H), 8.01 (t, J=6.0 Hz, 1H), 7.97 (d, J=8.1 Hz, 1H), 7.76-7.71(m, 1H), 7.69-7.65 (m, 1H), 7.32 (s, 1H), 7.22-7.14 (m, 2H), 7.09-7.01(m, 2H), 4.59-4.45 (m, 1H), 3.55-3.46 (m, 2H), 3.33-3.23 (m, 2H), 2.74(t, J=8.0 Hz, 2H), 2.42 (dd, J=14.4, 5.2 Hz, 1H), 2.37 (t, J=7.7 Hz,2H), 2.29 (dd, J=14.4, 8.3 Hz, 1H), 1.18 (s, 9H); ¹³C NMR (126 MHz,DMSO-d₆) δ 171.3, 171.0, 168.8, 165.4, 160.6 (d, J=241.1 Hz), 150.4,144.7, 137.9, 137.4, 132.4, 132.1, 129.9 (d, J=8.2 Hz), 129.2, 128.2,126.3, 121.5, 114.9 (d, J=20.1 Hz), 50.1, 50.0, 38.9, 38.8, 38.7, 36.9,30.1, 28.4. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −119.8 (m). HRMS calc. forC₂₉H₃₄FN₅O₄Na [M+Na]⁺: 558.2493. Found: 558.2484.

Example 54—Synthesis of PKS21259

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 4-fluoronaphthalene-1-carboxylic acid (9.6mg, 51 μmol) and PKS21249 (25.0 mg, 51 μmol). After completion of thereaction, the mixture was purified by preparative LCMS to give product(22.4 mg, 80%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.50 (t,J=5.7 Hz, 1H), 8.34-8.27 (m, 1H), 8.12-8.06 (m, 1H), 8.04-7.97 (m, 2H),7.71-7.62 (m, 3H), 7.41-7.31 (m, 2H), 7.21-7.13 (m, 2H), 7.09-7.00 (m,2H), 4.55-4.45 (m, 1H), 3.44-3.36 (m, 2H), 3.33-3.19 (m, 2H), 2.74 (t,J=7.9 Hz, 2H), 2.44 (dd, J=14.7, 6.1 Hz, 1H), 2.40-2.34 (m, 2H), 2.31(dd, J=14.7, 7.8 Hz, 1H), 1.17 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ171.3, 171.1, 168.9, 168.0, 160.6 (d, J=241.5 Hz), 158.6 (d, J=252.5Hz), 137.4, 131.5 (d, J=4.7 Hz), 131.2 (d, J=3.3 Hz), 129.9 (d, J=7.3Hz), 127.9, 127.0, 126.0 (d, J=9.1 Hz), 125.8, 122.8 (d, J=16.3 Hz),120.0 (d, J=5.5 Hz), 114.9 (d, J=20.5 Hz), 108.7 (d, J=19.9 Hz), 50.2,50.0, 38.9, 38.7, 38.6, 36.9, 30.1, 28.4; ¹⁹F NMR (471 MHz, DMSO-d₆) δ−119.8 (m), −122.6 (m). HRMS calc. for C₃₀H₃₄F₂N₄O₄Na [M+Na]⁺: 575.2446.Found: 575.2437.

Example 55—Synthesis of PKS21263

The title compound was synthesized by following the general procedurefor HATU mediated coupling of Boc-Asp-OBn (64.7 mg, 0.20 mmol) and1-(trifluoromethyl)cyclopropanamine (25.8 mg, 0.20 mmol). Aftercompletion of the reaction (2 hours), water was added and stirred at rtfor 30 minutes. The white precipitate formed was filtered, washed withwater and dried in air to give product (75 mg, 87%) as a white solid. ¹HNMR (500 MHz, Chloroform-d) δ 7.40-7.28 (m, 5H), 6.19-6.07 (m, 1H),5.79-5.61 (m, 1H), 5.19 (d, J=12.3 Hz, 1H), 5.15 (d, J=12.3 Hz, 1H),4.65-4.46 (m, 1H), 2.91-2.81 (m, 1H), 2.72 (dd, J=15.5, 3.9 Hz, 1H),1.42 (s, 9H), 1.32-1.25 (m, 2H), 1.07-1.00 (m, 2H).

Example 56—Synthesis of PKS21264

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21263 (70.0 mg, 0.163 mmol). After completionof the reaction, excess trifluoroacetic acid and dichloromethane wereevaporated. Crude was dried under vacuum and triturated with hexane.Hexane was decanted and colorless gum was dried under vacuum to giveproduct (72 mg, quant.). Product was used in next step without furtherpurification. ¹H NMR (500 MHz, DMSO-d₆) δ 9.00 (s, 1H), 8.39 (br, 3H),7.42-7.32 (m, 5H), 5.23-5.13 (m, 2H), 4.45-4.30 (m, 1H), 2.80-2.74 (m,2H), 1.29-1.17 (m, 2H), 1.01-0.86 (m, 2H).

Example 57—Synthesis of PKS21267

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-(4-fluorophenyl)propanoic acid (26.9 mg,0.160 mmol) and PKS21264 (71.1 mg, 0.160 mmol). After completion of thereaction, water was added and the mixture was stirred at roomtemperature for 30 minutes. The white precipitate formed was filtered,washed with water and dried in air to give product (70 mg, 91%) as awhite solid. ¹H NMR (500 MHz, Chloroform-d) δ 7.41-7.27 (m, 5H),7.17-7.08 (m, 2H), 6.99-6.87 (m, 2H), 6.70 (d, J=7.8 Hz, 1H), 6.12 (s,1H), 5.21-5.08 (m, 2H), 4.96-4.75 (m, 1H), 2.95-2.82 (m, 3H), 2.69-2.60(m, 1H), 2.57-2.42 (m, 2H), 1.33-1.21 (m, 2H), 1.05-0.91 (m, 2H).

Example 58—Synthesis of PKS21269

The title compound was synthesized by following the general procedurefor 0-debenzylation of PKS21267 (65.0 mg, 0.135 mmol). After completionof the reaction, the mixture was filtered through celite. Filtrate wasevaporated and dried to give product (52.8 mg, quant.) as a white solid.¹H NMR (500 MHz, DMSO-d₆) δ 12.60 (br, 1H), 8.78-8.64 (m, 1H), 8.14-8.02(m, 1H), 7.29-7.16 (m, 2H), 7.11-6.99 (m, 2H), 4.57-4.38 (m, 1H), 2.77(t, J=7.8 Hz, 2H), 2.54 (dd, J=15.3, 5.9 Hz, 1H), 2.45-2.33 (m, 3H),1.25-1.12 (m, 2H), 1.03-0.88 (m, 2H).

Example 59—Synthesis of PKS21273

The title compound was synthesized by following the general procedure ofHATU mediated coupling of PKS21269 (52.8 mg, 0.135 mmol) andN-Boc-ethylenediamine (23.8 mg, 0.149 mmol). After completion of thereaction, water was added and the mixture was stirred at roomtemperature for 30 minutes. The white precipitate formed was filtered,washed with water and dried in air to give product (70 mg, 97%) as awhite solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.59 (s, 1H), 8.01 (d, J=8.0Hz, 1H), 7.83 (t, J=5.6 Hz, 1H), 7.29-7.20 (m, 2H), 7.13-7.04 (m, 2H),6.78 (t, 1H), 4.59-4.45 (m, 1H), 3.15-2.90 (m, 4H), 2.79 (t, J=7.8 Hz,2H), 2.56-2.48 (m, 1H), 2.45-2.36 (m, 2H), 2.32 (dd, J=15.2, 7.7 Hz,1H), 1.39 (s, 9H), 1.24-1.16 (m, 2H), 1.02-0.93 (m, 2H).

Example 60—Synthesis of PKS21275

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21273 (65.0 mg, 0.122 mmol). After completionof the reaction (1.5 hours), excess trifluoroacetic acid anddichloromethane were evaporated. Crude was dried under vacuum andtriturated with diethyl ether to give a white solid. Diethyl ether wasdecanted and white solid was dried under vacuum to give product (62.3mg, 93%). The product was used in the next step without furtherpurification. ¹H NMR (500 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.10 (d, J=7.7Hz, 1H), 8.02 (t, J=6.0 Hz, 1H), 7.70 (br, 3H), 7.27-7.16 (m, 2H),7.14-7.01 (m, 2H), 4.55-4.42 (m, 1H), 3.36-3.27 (m, 1H), 3.27-3.18 (m,1H), 2.88-2.80 (m, 2H), 2.78 (t, J=8.0 Hz, 2H), 2.57-2.51 (m, 1H),2.44-2.32 (m, 3H), 1.24-1.14 (m, 2H), 1.01-0.89 (m, 2H).

Example 61—Synthesis of PKS21278

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 3-phenylbenzoic acid (5.5 mg, 28 μmol) andPKS21275 (13.7 mg, mol). The mixture was purified by preparative LCMS togive product (12.3 mg, 80%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆)δ 8.61 (s, 1H), 8.58 (t, J=5.6 Hz, 1H), 8.15-8.10 (m, 1H), 8.04 (d,J=8.1 Hz, 1H), 7.98 (t, J=5.7 Hz, 1H), 7.86-7.79 (m, 2H), 7.72 (d, J=7.6Hz, 2H), 7.54 (t, J=7.7 Hz, 1H), 7.48 (t, J=7.6 Hz, 2H), 7.39 (t, J=7.4Hz, 1H), 7.18 (dd, J=8.5, 5.6 Hz, 2H), 7.09-7.02 (m, 2H), 4.57-4.50 (m,1H), 3.30-3.15 (m, 4H), 2.74 (t, J=7.9 Hz, 2H), 2.55-2.50 (m, 1H),2.43-2.34 (m, 2H), 2.31 (dd, J=16.4, 8.8 Hz, 1H), 1.19-1.13 (m, 2H),0.94 (d, J=6.3 Hz, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 171.2, 170.9,170.4, 166.4, 160.6 (d, J=241.5 Hz), 140.2, 139.5, 137.4, 135.1, 129.9(d, J=8.5 Hz), 129.3, 129.0, 129.0, 127.8, 126.8, 126.4, 125.4 (q,J=275.9 Hz), 125.4, 114.9 (d, J=20.1 Hz), 49.8, 38.9, 38.7, 37.8, 36.9,31.8 (q, J=36.8 Hz), 30.0, 11.0, 10.9; 19F NMR (471 MHz, DMSO-d₆) δ−74.3 (s), −119.8 (m). HRMS calc. for C₃₂H₃₂F₄N₄O₄Na [M+Na]⁺: 635.2257.Found: 635.2267.

Example 62—Synthesis of PKS21279

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-5-(2-fluorophenyl)benzoic acid(6.4 mg, 28 μmol) and PKS21275 (13.7 mg, 25 μmol). The mixture waspurified by preparative LCMS to give product (13.2 mg, 81%) as a whitesolid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.59 (s, 1H), 8.43-8.33 (m, 1H), 8.02(d, J=8.1 Hz, 1H), 7.94 (t, J=5.7 Hz, 1H), 7.79 (dd, J=6.8, 2.4 Hz, 1H),7.72-7.66 (m, 1H), 7.56 (td, J=7.9, 1.9 Hz, 1H), 7.48-7.36 (m, 2H),7.35-7.27 (m, 2H), 7.18 (dd, J=8.5, 5.6 Hz, 2H), 7.08-7.01 (m, 2H),4.56-4.46 (m, 1H), 3.47-3.26 (m, 2H), 3.26-3.11 (m, 2H), 2.73 (t, J=7.8Hz, 2H), 2.55-2.50 (m, 1H), 2.41-2.25 (m, 3H), 1.19-1.10 (m, 2H),0.96-0.88 (m, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 171.2, 170.9, 170.3,163.6, 160.6 (d, J=241.6 Hz), 159.8 (d, J=246.4 Hz), 158.8 (d, J=251.4Hz), 137.4, 132.7 (d, J=6.0 Hz), 131.3, 130.8, 130.4, 130.0 (d, J=8.8Hz), 129.9 (d, J=7.2 Hz), 126.6 (d, J=14.4 Hz), 125.4 (q, J=275.8 Hz),125.3-124.9 (m), 124.2 (d, J=14.6 Hz), 116.5 (d, J=22.1 Hz), 116.2 (d,J=21.9 Hz), 114.9 (d, J=21.6 Hz), 49.8, 38.9, 38.5, 37.7, 36.8, 31.7 (q,J=37.1 Hz), 30.0, 11.0, 10.9; ¹⁹F NMR (471 MHz, DMSO-d₆) δ −74.4 (s),−117.8 (m), −119.8 (m), −120.8 (m). HRMS calc. for C₃₂H₃₀F₆N₄O₄Na[M+Na]⁺: 671.2069. Found: 671.2053.

Example 63—Synthesis of PKS21270

The title compound was synthesized by following the general procedurefor HATU mediated coupling of 2-fluoro-5-(2-fluorophenyl)benzoic acid(117.10 mg, 500 μmol) and N-boc-ethylenediamine (88.12 mg, 550 μmol).After completion of the reaction, water was added and the mixture wasstirred at room temperature for 30 minutes. The white precipitate formedwas filtered, washed with water, and dried in air to give product (160.0mg, 85%) as a white solid. ¹H NMR (500 MHz, Chloroform-d) δ 8.22 (d,J=7.3 Hz, 1H), 7.69-7.62 (m, 1H), 7.48-7.40 (m, 1H), 7.36-7.29 (m, 1H),7.25-7.10 (m, 4H), 4.96 (br, 1H), 3.64-3.58 (m, 2H), 3.43-3.37 (m, 2H),1.42 (s, 9H).

Example 64—Synthesis of PKS21274

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21270 (150.0 mg, 399 μmol). After completionof the reaction, excess trifluoroacetic acid and dichloromethane wereevaporated. Crude was dried under vacuum and triturated with diethylether to give a white solid. Diethyl ether was decanted and white solidwas dried under vacuum to give product (155 mg, quant.). The product wasused in the next step without further purification. ¹H NMR (500 MHz,DMSO-d₆) δ 8.61-8.50 (m, 1H), 8.04-7.76 (m, 4H), 7.76-7.67 (m, 1H),7.61-7.51 (m, 1H), 7.49-7.39 (m, 2H), 7.38-7.30 (m, 2H), 3.59-3.46 (m,2H), 3.07-2.93 (m, 2H).

Example 65—Synthesis of PKS21277

The title compound was synthesized by following the general procedurefor HATU mediated coupling of(2S)-2-(tert-butoxycarbonylamino)-4-(tert-butylamino)-4-oxo-butanoicacid (28.8 mg, 100 μmol) and PKS21274 (39.0 mg, 100 μmol). Aftercompletion of the reaction, the mixture was purified by preparative LCMSto give product (43.0 mg. 79%) as a white solid. ¹H NMR (500 MHz,DMSO-d₆) δ 8.44-8.33 (m, 1H), 7.98 (t, J=5.6 Hz, 1H), 7.83-7.75 (m, 1H),7.74-7.67 (m, 1H), 7.60-7.52 (m, 1H), 7.48-7.42 (m, 1H), 7.42-7.29 (m,4H), 6.74 (d, J=8.2 Hz, 1H), 4.24-4.13 (m, 1H), 3.40-3.24 (m, 3H),3.24-3.15 (m, 1H), 2.38 (dd, J=14.3, 5.4 Hz, 1H), 2.30 (dd, J=14.3, 8.4Hz, 1H), 1.34 (s, 9H), 1.19 (s, 9H). HRMS calc. forC₂₈H₃₆F₂N₄O₅Na[M+Na]⁺: 569.2551. Found: 569.2564.

Example 66—Synthesis of PKS21284

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21277 (21.0 mg, 38 μmol). After completion ofthe reaction, excess trifluoroacetic acid and dichloromethane wereevaporated. Crude was purified by preparative LCMS to give product (19.0mg, 88%) as a colorless gum. ¹H NMR (500 MHz, DMSO-d₆) δ 8.49 (t, J=5.2Hz, 1H), 8.46-8.41 (m, 1H), 8.10 (d, J=4.8 Hz, 3H), 7.82-7.78 (m, 2H),7.73-7.69 (m, 1H), 7.59-7.54 (m, 1H), 7.49-7.39 (m, 2H), 7.36-7.30 (m,2H), 4.00-3.94 (m, 1H), 3.43-3.29 (m, 3H), 3.29-3.19 (m, 1H), 2.65 (dd,J=16.5, 5.1 Hz, 1H), 2.55 (dd, J 16.5, 7.8 Hz, 1H), 1.22 (s, 9H).

Example 67—Synthesis of PKS21293

To a solution of PKS21284 (10.7 mg, 19 μmol) in dichloromethane (1.00mL), triethylamine (5.77 mg, 57.00 μmol, 7.90 μL) was added at 0° C. Thesolution was warmed to room temperature (over 15 minutes) andcyclopropanesulfonyl chloride (5.3 mg, 38 μmol) was added in oneportion. The mixture was stirred at room temperature overnight. Aftercompletion of the reaction, dichloromethane was evaporated and crude waspurified by preperative LCMS to give product (9.2 mg, 88%) as a whitesolid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.40-8.35 (m, 1H), 8.12 (t, J=5.6 Hz,1H), 7.80-7.76 (m, 1H), 7.72-7.67 (m, 1H), 7.57 (td, J=7.9, 1.7 Hz, 1H),7.49-7.37 (m, 3H), 7.36-7.29 (m, 3H), 4.14-4.03 (m, 1H), 3.42-3.25 (m,3H), 3.25-3.16 (m, 1H), 2.49-2.42 (m, 2H), 2.37 (dd, J=14.8, 7.4 Hz,1H), 1.19 (s, 9H), 0.89-0.78 (m, 4H). ¹³C NMR (126 MHz, DMSO-d₆) δ171.2, 168.4, 163.5, 159.0 (d, J=247.3 Hz), 158.8 (d, J=251.1 Hz),132.9-132.5 (m), 131.3 (d, J=2.6 Hz), 130.8, 130.3, 130.0 (d, J=7.4 Hz),126.6 (d, J=12.8 Hz), 125.1 (d, J=2.9 Hz), 124.2 (d, J=14.6 Hz), 116.5(d, J=22.9 Hz), 116.2 (d, J=22.1 Hz), 53.8, 50.1, 39.8, 39.0, 38.4,30.2, 28.4, 5.0, 4.8. ¹⁹F NMR (471 MHz, DMSO-d₆) δ −117.8 (m), −120.8(m).

Example 68—Synthesis of PKS21294

Hunig's base (8.7 mg, 67 μmol, 11.7 μL) and N,N-dimethylpyridin-4-amine(1.4 mg, 11 μmol) were added to a solution of PKS21284 (12.5 mg, 22μmol) in dichloromethane (1.00 mL) at 0° C. The solution was stirred for5 minutes, and acetic anhydride (2.7 mg, 26.8 μmol, 2.5 μL) was added.The reaction mixture was stirred at 0° C. for 1 hour. After completionof the reaction, dichloromethane was evaporated and crude was purifiedby preparative LCMS to give product (7.1 mg, 65%) as a white solid. ¹HNMR (500 MHz, DMSO-d₆) δ 8.43-8.33 (m, 1H), 7.98 (t, J=5.7 Hz, 1H), 7.95(d, J=8.1 Hz, 1H), 7.81-7.77 (m, 1H), 7.72-7.67 (m, 1H), 7.57 (td,J=7.9, 1.7 Hz, 1H), 7.48-7.42 (m, 1H), 7.39 (dd, J=10.2, 8.6 Hz, 1H),7.36-7.30 (m, 3H), 4.50-4.39 (m, 1H), 3.41-3.28 (m, 2H), 3.28-3.12 (m,2H), 2.42 (dd, J=14.5, 5.8 Hz, 1H), 2.29 (dd, J=14.5, 8.1 Hz, 1H), 1.80(s, 3H), 1.18 (s, 9H); ¹³C NMR (126 MHz, DMSO-d₆) δ 171.4, 169.0, 168.9,163.6, 159.0 (d, J=246.9 Hz), 158.8 (d, J=252.6 Hz), 132.8-132.5 (m),131.4-131.2 (m), 130.8, 130.3, 130.0 (d, J=7.5 Hz), 126.6 (d, J=12.8Hz), 125.0 (d, J=2.5 Hz), 124.2 (d, J=14.5 Hz), 116.5 (d, J=22.1 Hz),116.2 (d, J=21.9 Hz), 50.2, 50.0, 39.0, 38.7, 38.5, 28.4, 22.6; ¹⁹F NMR(471 MHz, DMSO-d₆) δ −117.9 (m), −120.8 (m).

Example 69—Synthesis of PKS21276

The title compound was synthesized by following the general procedurefor HATU mediated coupling of Boc-glycine (19.3 mg, 110 μmol) andPKS21274 (39.0 mg, 100 μmol). After completion of the reaction, themixture was purified by preparative LCMS to give product (38.0 mg, 88%)as a colorless solid. ¹H NMR (500 MHz, Chloroform-d) δ 8.23-8.16 (m,1H), 7.69-7.62 (m, 1H), 7.47-7.40 (m, 1H), 7.37-7.31 (m, 1H), 7.26-7.11(m, 4H), 6.89 (t, J=5.7 Hz, 1H), 5.16 (br, 1H), 3.80 (d, J=4.2 Hz, 2H),3.67-3.61 (m, 2H), 3.57-3.51 (m, 2H), 1.41 (s, 9H).

Example 70—Synthesis of PKS21285

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21276 (32.0 mg, 74 μmol). After completion ofthe reaction, excess trifluoroacetic acid and dichloromethane wereevaporated. Crude was dried under vacuum to give a product (33 mg,quant.). The product was used in next step without further purification.10 ¹H NMR (500 MHz, DMSO-d₆) δ 8.52-8.43 (m, 2H), 8.09-7.98 (m, 3H),7.82-7.77 (m, 1H), 7.74-7.68 (m, 1H), 7.57 (td, J=7.9, 1.7 Hz, 1H),7.48-7.38 (m, 2H), 7.37-7.30 (m, 2H), 3.56-3.49 (m, 2H), 3.41-3.34 (m,2H), 3.34-3.28 (m, 2H).

Example 71—Synthesis of PKS21289

The title compound was synthesized by following the general procedurefor sulfonamide preparation of PKS21285 (16.00 mg, 35.77 μmol) with4-methylbenzenesulfonyl chloride (13.6 mg, 72 μmol). After completion ofthe reaction, dichloromethane was evaporated and crude was purified bypreparative LCMS to give product (14.8 mg, 85%) as a white solid. ¹H NMR(500 MHz, DMSO-d₆) δ 8.43-8.34 (m, 1H), 8.02 (t, J=5.8 Hz, 1H), 7.86 (s,1H), 7.80-7.74 (m, 1H), 7.73-7.69 (m, 1H), 7.67 (d, J=8.3 Hz, 2H), 7.57(td, J=7.9, 1.9 Hz, 1H), 7.49-7.41 (m, 1H), 7.44-7.28 (m, 5H), 3.43-3.31(m, 2H), 3.31-3.23 (m, 2H), 3.23-3.15 (m, 2H), 2.37 (s, 3H). ¹³C NMR(126 MHz, DMSO-d₆) δ 167.8, 163.5, 159.0 (d, J=246.9 Hz), 158.8 (d,J=251.2 Hz), 142.8, 137.1, 132.7 (d, J=8.7 Hz), 131.3, 130.8, 130.3,130.0 (d, J=8.5 Hz), 129.5, 126.7, 126.6-126.5 (m), 125.1, 124.2 (d,J=14.6 Hz), 116.5 (d, J=22.4 Hz), 116.2 (d, J=22.1 Hz), 45.3, 39.0,38.2, 21.0; ¹⁹F NMR (471 MHz, DMSO-d₆) δ −117.9 (m), −120.9 (m).

Example 72—Synthesis of PKS21280

Triethylamine (75.8 mg, 749 μmol, 104 μL) was added to a solution ofPKS21274 (39.0 mg, 100 μmol) in dichloromethane (3.00 mL) at 0° C. Themixture was stirred for 10 minutes and Boc-Ala-OSu (31.5 mg, 110 μmol)was added. The reaction mixture was allowed to warm to room temperatureslowly. After completion of the reaction (2 hours), dichloromethane wasevaporated and crude was purified by preparative LCMS to give product(36.3 mg, 81%) as a white solid. ¹H NMR (500 MHz, DMSO-d6) δ 8.36 (t,J=5.5 Hz, 1H), 7.92 (t, J=5.5 Hz, 1H), 7.78 (dd, J=7.2, 2.4 Hz, 1H),7.73-7.66 (m, 1H), 7.59-7.53 (m, 1H), 7.48-7.42 (m, 1H), 7.40 (dd,J=10.3, 8.5 Hz, 1H), 7.36-7.30 (m, 2H), 6.84 (d, J=7.4 Hz, 1H),3.96-3.74 (m, 1H), 3.46-3.13 (m, 4H), 1.34 (s, 9H), 1.15 (d, J=7.2 Hz,3H).

Example 73—Synthesis of PKS21286

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21280 (30.0 mg, 67 μmol). After completion ofthe reaction, excess trifluoroacetic acid and dichloromethane wereevaporated. Crude was dried under vacuum to give product (31.0 mg,quant.). The product was used in the next step without furtherpurification. ¹H NMR (500 MHz, DMSO-d₆) δ 8.50 (t, J=5.9 Hz, 1H), 8.45(t, J=5.1 Hz, 1H), 8.20-7.99 (m, 3H), 7.78 (dd, J=6.8, 2.3 Hz, 1H),7.74-7.67 (m, 1H), 7.60-7.52 (m, 1H), 7.49-7.38 (m, 2H), 7.37-7.29 (m,2H), 3.84-3.71 (m, 1H), 3.46-3.30 (m, 3H), 3.30-3.14 (m, 1H), 1.34 (d,J=7.0 Hz, 3H).

Example 74—Synthesis of PKS21290

The title compound was synthesized by following the general procedurefor sulfonamide preparation of PKS21286 (16.0 mg, 35 μmol) with4-methylbenzenesulfonyl chloride (9.9 mg, 52 μmol). After completion ofthe reaction, dichloromethane was evaporated and the crude was purifiedby preparative LCMS to give product (12.4 mg, 71%) as a white solid. ¹HNMR (500 MHz, DMSO-d₆) δ 8.36-8.30 (m, 1H), 7.99 (t, J=5.7 Hz, 1H), 7.89(br, 1H), 7.78-7.75 (m, 1H), 7.72-7.68 (m, 1H), 7.65 (d, J=8.3 Hz, 2H),7.56 (td, J=7.9, 1.9 Hz, 1H), 7.48-7.38 (m, 2H), 7.37-7.29 (m, 4H),3.70-3.60 (m, 1H), 3.27-3.01 (m, 4H), 2.36 (s, 3H), 1.03 (d, J=7.1 Hz,3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 171.5, 163.4, 159.0 (d, J=245.7 Hz),158.8 (d, J=252.8 Hz), 142.6, 138.1, 132.7 (d, J=7.8 Hz), 131.3, 130.8,130.3, 130.0 (d, J=8.7 Hz), 129.4, 126.6, 126.6 (d, J=12.1 Hz), 125.1(d, J=2.9 Hz), 124.2 (d, J=14.6 Hz), 116.5 (d, J=22.0 Hz), 116.2 (d,J=23.5 Hz), 52.0, 38.9, 38.1, 21.0, 18.8. ¹⁹F NMR (471 MHz, DMSO-d₆) δ−117.8 (m), −120.9 (m).

Example 75—Synthesis of PKS21281

The title compound was synthesized by following the general procedurefor HATU mediated coupling of Boc-Asn-OH (23.2 mg, 100 μmol) andPKS21274 (39.0 mg, 100 mol). After completion of the reaction, themixture was purified by preparative LCMS to give product (43.6 mg, 89%)as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 8.36 (t, J=5.6 Hz, 1H),7.95 (t, J=5.6 Hz, 1H), 7.79 (dd, J=7.1, 2.5 Hz, 1H), 7.70 (ddt, J=8.7,4.4, 2.0 Hz, 1H), 7.57 (td, J=7.9, 1.7 Hz, 1H), 7.49-7.41 (m, 1H), 7.40(dd, J=10.3, 8.6 Hz, 1H), 7.38-7.28 (m, 2H), 7.28-7.21 (m, 1H), 6.87 (s,1H), 6.81 (d, J=8.0 Hz, 1H), 4.18 (td, J=8.0, 5.3 Hz, 1H), 3.37-3.22 (m,3H), 3.19 (dq, J=12.6, 6.3 Hz, 1H), 2.43 (dd, J=15.0, 5.3 Hz, 1H), 2.35(dd, J=15.0, 8.1 Hz, 1H), 1.34 (s, 9H).

Example 76—Synthesis of PKS21283

The title compound was synthesized by following the general procedurefor Boc-deprotection of PKS21281 (30.0 mg, 61 μmol). After completion ofthe reaction, excess trifluoroacetic acid and dichloromethane wereevaporated. Crude was dried under vacuum and triturated with diethylether to give a white solid. Diethyl ether was decanted and the whitesolid was dried under vacuum to give product (30.8 mg, quant.). Theproduct was used in the next step without further purification. ¹H NMR(500 MHz, DMSO-d₆) δ 8.54 (t, J=5.3 Hz, 1H), 8.49-8.41 (m, 1H), 8.11 (d,J=5.2 Hz, 3H), 7.84-7.76 (m, 1H), 7.74-7.69 (m, 1H), 7.65 (br, 1H), 7.57(td, J=7.9, 1.7 Hz, 1H), 7.48-7.38 (m, 2H), 7.37-7.28 (m, 2H), 7.23 (br,1H), 4.05-3.95 (m, 1H), 3.43-3.29 (m, 3H), 3.29-3.20 (m, 1H), 2.70 (dd,J=16.8, 4.6 Hz, 1H), 2.58 (dd, J=16.8, 8.3 Hz, 1H).

Example 77—Synthesis of PKS21288

The title compound was synthesized by following the general procedurefor sulfonamide preparation of PKS21283 (15.1 mg, 30 μmol) with4-methylbenzenesulfonyl chloride (11.4 mg, 60 μmol). After completion ofthe reaction, dichloromethane was evaporated and crude was purified bypreperative LCMS to give product (13.6 mg, 83%) as a white solid. ¹H NMR(500 MHz, DMSO-d₆) δ 8.33-8.24 (m, 1H), 7.97 (t, J=5.8 Hz, 1H), 7.85(br, 1H), 7.78 (dd, J=7.1, 2.4 Hz, 1H), 7.74-7.68 (m, 1H), 7.64 (d,J=8.3 Hz, 2H), 7.57 (td, J=7.9, 1.9 Hz, 1H), 7.48-7.38 (m, 2H),7.36-7.28 (m, 4H), 7.27 (d, J=2.3 Hz, 1H), 6.84 (d, J=2.3 Hz, 1H),4.04-3.95 (m, 1H), 3.25-3.09 (m, 2H), 3.09-2.92 (m, 2H), 2.39-2.30 (m,4H), 2.21 (dd, J=15.1, 6.8 Hz, 1H); ¹³C NMR (126 MHz, DMSO-d₆) δ 170.7,170.1, 163.4, 159.0 (d, J=246.9 Hz), 158.8 (d, J=251.2 Hz), 142.5,138.1, 132.7 (d, J=8.3 Hz), 131.3, 130.8, 130.3, 130.0 (d, J=7.8 Hz),129.2, 126.7, 126.6, 125.3-124.9 (m), 124.1 (d, J=14.1 Hz), 116.6 (d,J=23.4 Hz), 116.2 (d, J=22.1 Hz), 53.4, 38.8, 38.2, 38.2, 21.0; ¹⁹F NMR(471 MHz, DMSO-d₆) δ −117.8 (m), −120.9 (m).

Example 78—Determination of IC₅₀ Values Against Human Constitutive andImmuno-Proteasomes

A 96-well-plate assays were used to determine the IC_(50s) against thechymotryptic β5 activities of the proteasomes. Both human constitutiveproteasome and immunoproteasome were purchased from Boston Biochem Inc.1 μL of 100× compound in DMSO at designated concentrations were spottedat the bottom of the wells. DMSO was used as a control. Finalconcentrations of the inhibitors were from 100 μM to 0.098 μM. Thehydrolysis of the substrate over time in each well was monitored atexcitation 360 nm, emission 460 nm for 90 minutes. IC_(50s) wereestimated by fitting the velocities of hydrolysis against compoundconcentrations using PRISM. For IC_(50s) against human proteasomes, theconcentrations were 0.25 nM for the c-20S, 0.4 nM for the i-20S.Suc-LLVY-AMC was used for 35c at final concentration of 25 μM, andAc-ANW-AMC for b5i at final concentration of 15 μM. SDS (0.02%) was usedas activator, and 0.01% BSA was used in the reaction buffer.

Example 79—Determination of EC₅₀ Values Against Plasmodium Falciparum

Continuous in vitro cultures of Plasmodium falciparum: were maintainedin human red blood cells (RBC) diluted to a hematocrit of 5% in RPMI1640 medium with HEPES and Hypoxanthine and completed with 0.5% AlbumaxII (Invitrogen), 0.25% sodium bicarbonate, and 0.1 mg/ml gentamicin.Parasites were incubated at 37° C. in a gas mixture of 5% oxygen, 5%carbon dioxide, and 90% nitrogen.

Parasite sensitivity to novel compounds were determined using a SYBRGreen (Invitrogen S1046) drug assay protocol. P. falciparum of variousclinic stains were cultured with compounds at concentrations in a seriesof dilution in a clear, sterile 96-well plate for 72 hours.Subsequently, 150 l of the cultures were transferred to a black 96-wellplate and placed in the freezer for red blood cell lysis. The plateswere thawed and suspended in a Sybr Green Lysis buffer. GraphPad Prismwas used to analyze the raw data collected through the super old platereader in the back of the lab. Drug concentrations were converted tologarithms, normalized, and then curve fitted by non-linear regressionto obtain EC₅₀ values. The results are shown in Tables 1 and 2.

TABLE 1 EC50s of Selected Compounds Against the Growth of Plasmodiumfalciparum in Red Blood Cells. EC₅₀ (nM) ID (P.f.)¹ PKS3080 23 PKS30814520 PKS21003 >2770 PKS21004 4.6 PKS21018 364 PKS21019 30 PKS21025 320PKS21026 100 PKS21221 0.55 PKS21229 1 PKS21224 11 PKS21291 11 PKS212922.9 PKS21287 34 TDI4258 29 ¹P.f.: 3D7, IC50s were determined as reported(Heinberg et al., “Direct Evidence for the Adaptive Role of Copy NumberVariation on Antifolate Susceptibility in Plasmodium Falciparum,” Mol.Microbiol. 88: 702-712 (2013), which is hereby incorporated by referencein its entirety).

TABLE 2 EC_(50s) of PKS21004 and PKS21003 Against P. falciparum Strains.EC50s (nM) P. f. Strain Phenotype PKS21004 PKS21003 3D7 SUL - R0.0046 >2.77 HB3 PYR - R 0.010 >2.77 D6 Pan - S 0.004 >2.77 Sb1-a6ATOV - R 0.0048 >2.77 Dd2 MDR 0.0048 >2.77 V1S MDR 0.0017 >2.77 IPC 3663ART - S 0.006 >2.77 IPC 5188 ART - S 0.0015 >2.77 IPC 4884 ART - R0.0052 >2.77 IPC 5202 ART - R 0.0046 NT IPC 4912 ART - R 0.0058 NT ART:artemisinin; ATOV: Atovaquone; MDR: multi-drug resistant; PYR:pyrimethamine; R: resistant; S: sensitive; SUL: sulfadoxine.

Example 80—Cell Viability Assay

Multiple myeloma cell lines MM. 1S (200,000 cells/mL) and RPMI 8226(200,000 cells/mL), B lymphoma cell line Karpas 106P (800,000 cells/mL),and liver cancer cell line HepG2 (12,000 cells/mL) were used todetermine the cytotoxicity of compounds. Cells were cultured at 37° C.in a humidified air/5% CO₂ atmosphere in medium supplemented with 10%fetal bovine serum, except for the medium for Karpas-1106P cells whichcontained 20% fetal bovine serum, and 100 units/ml penicillin/100 μg/mlstreptomycin in RPMI 1640 medium. 12,000 cells/well. Cells plated in a96-well plate were treated with compounds at the indicatedconcentrations for 72 hours at 37° C. in a tissue culture incubator with5% CO₂. Viable cells were counted using Cell-titer/Glo™ assay kit. EC50swere calculated using PRISM (Graphpad). The results are shown in Table3.

TABLE 3 Inhibition of Intracellular Proteasomal Activities andCytotoxicity of PKS21221. IC50 (μM) Karpas EC50 (μM) Karpas β5i β5 HepGβ5c MM1.S 8226 Karpas HepG2 0.172 0.118 2.0 0.095 0.075 0.84 3.0 β5iactivity was assayed with (Ac-ANW)2-R110; β5 and β5c activity wasassayed with suc-LLVY-luciferin. Data were given as mean ± SEM.

Example 81—Protocol for Anti-Malarial Assay

Plasmodium falciparum: parasites were cultured in human red cells. Drugassays were run in 96 well plates at a total volume of 200 μl. Assayswere set up at a starting parasitemia (percent of infected red cells) of0.5%. Test compounds were plated at concentrations of 2778 nM to 0.4 nM.The test plates were grown under standard low oxygen conditions at 37°C. for 72 hours. At that time the plates were prepared for growthdetermination, first the plates were frozen and thawed to lyse the redblood cells, 150 l of the lysed thawed culture was transferred to ablack 96 well plate, and mixed in a lysis buffer with SYBR green DNAdye. The fluorescence of each well was recorded in a plate reader withexcitation wavelength 490 nm and emission wavelength 530 nm andnormalized relative to DMSO control. The EC50s were determined usingPrism software.

Asexual replication of NF54 peg4-tdTomato parasites was eliminated bytreating with 50 mM GlcNAc and 20 U/mL Heparin for 3 days. NF54peg4-tdTomato parasites then were maintained using standard culturetechniques. Gametocytes were induced synchronously according to Fivelmanet al., “Improved Synchronous Production of Plasmodium FalciparumGametocytes in Vitro,” Mol, Biochem. Parasitol. 154(1):119-123 (2007),which is hereby incorporated by reference in its entirety. Asexualreplication was eliminated by treating with 50 mM GlcNAc for 3 days.Gametocyte killing assays were setup on days 5 and 10 in triplicate96-well format at 1% hematocrit and 2% gametocytemia. Compounds weresetup in triplicate and serially diluted 3-fold. In addition, 6 solventcontrols (DMSO) were distributed evenly across the plate. Following a 72hour incubation, Stage III-IV (Day 5-7) and Stage IV-V (Day 10-12),(start the incubation in the afternoon on Day 5 and Day 10, take outplate for assay on Day8 and Day13 afternoon) cultures were stained with16 nM Hoechst 33342 and 50 nM DilC1(5) for 30 min at 37 C. Using a CytekDxP12 flow cytometer, gametocytemia was determined by gating for DNA+,hemozoin-high cells and gametocyte viability was inferred based onmitochondrial membrane potential-dependent accumulation of DilC1(5) for2000-3000 gametocytes (Tanaka et al., “Potent Plasmodium FalciparumGametocytocidal Activity of Diaminonaphthoquinones, Lead AntimalarialChemotypes Identified in an Antimalarial Compound Screen,” Antimicrob.Agents Chemother. 2015, 59:1389 (2015), which is hereby incorporated byreference in its entirety). Mean DilC1(5) signal was normalized tosolvent control and the overall minimum and used to calculate the EC50(FIG. 3 and Table 4).

TABLE 4 EC50s of Compounds Against Pf Gametocytes. Compound ID EC₅₀PKS21004  50 nM PKS21224 162 nM PKS21287 206 nM

FIG. 4A shows accumulation of poly-ub proteins in P. falciparumschizonts 4 hours post treatment with inhibitors at indicatedconcentrations (BTZ: bortezomib). FIG. 4B shows that AsnEDAsspecifically inhibit 35 active subunit of Pf20S. Pf lysates wereincubated with inhibitors at indicated concentrations for 1 hour priorto incubation with MV151 (2 μM) for an additional hour. SDS pages werescanned on a Typhoon fluorescent scanner. Top: PKS21004 inhibited thelabeling of Pf20S J35, whereas PKS21003 did not. BTZ was used as apositive control. Middle and bottom: PKS21004 and PKS21287dose-dependently inhibited the labeling of the Pf20S β5. FIG. 4C showsdose-dependent killing of P. berghei on sporozoite stage by PKS21004(P/T: sporozoites pre-treated with PKS21004 for 30 minutes on ice, andadded to HepG2 cells in 10× media volume and the media was replacedafter 4 hours (triangle). PKS21004 & sporozoites incubated with HepG2and media was replaced after 6 hours (square) and 14 hours (circle).EC50s were 157 nM, 41 nM, and 21 nM, respectively.

Example 82—Results and Discussion

To regulate immune responses through proteasome inhibition with lessmechanism-based toxicity to immune cells and little or none to othercells, it would be useful to inhibit i-20S selectively, sparing c-20S.Consistent with this notion, and unlike disruption of genes encodingc-20S subunits, disruption of genes encoding β1i, β2i, and β5i resultsin mice that are healthy, fertile, and immunocompetent (Kincaid et al,“Mice Completely Lacking Immunoproteasomes Show Major Changes in AntigenPresentation,” Nat. Immunol. 13:129-135 (2012), which is herebyincorporated by reference in its entirety). Indeed, relatively selectiveinhibition of β5i over β5c with the compound ONX-0914 has beenefficacious in several mouse models of autoimmune disease (Kalim et al.,“Immunoproteasome Subunit LMP7 Deficiency and Inhibition Suppresses Th1and Th17 but Enhances Regulatory T Cell Differentiation,” J. Immunol.189:4182-4193 (2012); Basler et al., “Inhibition of the ImmunoproteasomeAmeliorates Experimental Autoimmune Encephalomyelitis,” EMBO Mol Med6:226-238 (2014); Basler et al., “Prevention of Experimental Colitis bya Selective Inhibitor of the Immunoproteasome,” J. Immunol. 185:634-641(2010); Muchamuel et al., “A Selective Inhibitor of the ImmunoproteasomeSubunit LMP7 Blocks Cytokine Production and Attenuates Progression ofExperimental Arthritis,” Nat. Med. 15:781-787 (2009); Ichikawa et al.,“Beneficial Effect of Novel Proteasome Inhibitors in Murine Lupus viaDual Inhibition of Type I Interferon and Autoantibody-Secreting Cells,”Arthritis Rheum, 64:493-503 (2012), which are hereby incorporated byreference in their entirety). However, ONX-0914 belongs to the peptideepoxyketone class of inhibitors whose irreversible mechanism involvesrecruiting the hydroxyl and amino groups of the active site Thr^(1N)into formation of a morpholine adduct with the epoxyketone warhead(Groll et al., “Crystal Structure of Epoxomicin: 20S Proteasome Revealsa Molecular Basis for Selectivity of α′,β′-Epoxyketone ProteasomeInhibitors,” J. Am. Chem. Soc. 122:1237-1238 (2000), which is herebyincorporated by reference in its entirety). Long-term use of anirreversible inhibitor presents a risk of toxicity from the gradual,cumulative inhibition of c-20S and unknown targets. Therefore, it wouldbe desirable to develop inhibitors that are both more highly selectivefor i-20S and reversible. An additional benefit might accrue from anoncompetitive mode of action, so that progressive accumulation ofsubstrate does not lessen the degree of inhibition. The presentapplication reports the serendipitous discovery of a novel class ofnoncovalent compounds that non-competitively and selectively inhibitchymotryptic β5i over β5c.

A novel class of the N,C-capped dipeptides that selectively inhibit theMycobacterium tuberculosis proteasome over human c-20S was recentlyreported (Lin et al., “Inhibitors Selective for Mycobacterial VersusHuman Proteasomes,” Nature 461(7264):621-626 (2009), which is herebyincorporated by reference in its entirety). It was later found that thisclass of inhibitors also selectively inhibits i-20S over c-20S (Fan etal., “Oxathiazolones Selectively Inhibit the Human Immunoproteasome overthe Constitutive Proteasome,” ACS Med. Chem. Lett. 5:405-410 (2014),which is hereby incorporated by reference in its entirety), reflectingthat the mycobacterial and human c-20S proteasomes share an enlarged S1pocket and preferred oligopeptide substrates (Lin et al., “DistinctSpecificities of Mycobacterium Tuberculosis and Mammalian Proteasomesfor N-Acetyl Tripeptide Substrates,” J. Biol. Chem. 283:34423-34431(2008); Blackburn et al., “Characterization of a New Series ofNon-Covalent Proteasome Inhibitors with Exquisite Potency andSelectivity for the 20S β5-Subunit,” Biochem. J. 430:461-476 (2010),which are hereby incorporated by reference in their entirety). A novelclass of N,C-capped dipeptidomimetics was developed by incorporatingβ-amino acid into the N,C-capped dipeptides with marked selectivity fori-20S over c-20S (Singh et al., “Immunoproteasome β5i-SelectiveDipeptidomimetic Inhibitors,” Chem. Med. Chem. 11(19):2127-2131 (2016),which is hereby incorporated by reference in its entirety). Thus, someof the features found in the mycobacterial 20S inhibitors were leveragedfor the rational design of i-20S selective inhibitors. The amide bondsin select N,C-capped dipeptides were systematically replaced withbioisosteres. Reversing the amide bond at the C-cap and replacing theamino acid with an aromatic carboxyl acid resulted in a novel chemotype:Asn-ethylenediamine (AsnEDA). The first compound of this class, PKS3080,yielded modest IC50s of 0.37 and 1.22 μM against human β5i and 05c,respectively. Replacement of the 1-naphthoyl with 2-naphthoyl (producingPKS21025) improved potency against β5i by 3-fold with increasedselectivity to 26-fold.

Next, the N-cap, C-cap, ethylene, and Asn side chain substitutions werevaried. Replacement of the N-cap with [1,1′-biphenyl]-4-carboxamide(PKS21003) and [1,1′-biphenyl]-3-carboxamide (PKS21004) drasticallyimpacted activity. PKS21003 had no detectable activity against eitherβ5i or β5c (IC50>100 μM for both), whereas PKS21004 was a potentinhibitor of both β5i and β5c, with IC50s of 0.058 and 0.326 μM,respectively. Inhibition of the β5 subunits was specific, as noinhibition was observed of β1 or β2 activities in either i-20S or c-20S(Table 5).

TABLE 5 IC50s of compounds against human immunoproteasome β5i andconstitutive proteasome β5c subunits. IC50 (μM) Hu i-20S Hu c-20S IDStructures (Ac-ANW-AMC) (Suc-LLVY-AMC) PKS3080

0.37 1.22 PKS21003

>100 >100 PKS21004

0.058 0.326 PKS21018

2.35 14.6 PKS21019

0.276 3.46 PKS21025

0.284 3.60 PKS21026

0.11 1.16 PKS21028

2.06 7.25 PKS21030

0.39 0.068 PKS21186

0.125 2.76 PKS21187

0.016 0.36 PKS21195

0.059 1.94 PKS21196

0.044 0.818 PKS21208

0.027 1.04 PKS21221

0.0041 0.106 PKS21224

0.746 33.74 PKS21225

0.269 11.76 PKS21228

0.012 0.42 PKS21229

0.019 0.51 PKS21250

65.6 >100 PKS21251

6.34 39 PKS21254

7.77 57.4 PKS21255

0.74 3.64 PKS21258

2.41 22.66 PKS21259

0.158 2.24 PKS21276

24.25 >100 PKS21277

0.112 3.65 PKS21280

33.23 >100 PKS21281

>100 >100 PKS21278

0.080 0.165 PKS21279

0.037 0.114 PKS21282

0.069 1.29 PKS21284

0.626 3.09 PKS21287

1.15 21.06 PKS21288

4.14 >100 PKS21289

40.3 60.8 PKS21290

>100 >100 PKS21291

0.055 0.52 PKS21292

0.015 0.187 PKS21293

0.015 0.96 PKS21294

0.62 13.3 PKS21295

0.129 2.39 PKS21315

0.174 N/A

A washout experiment was used to confirm the reversibility of this classof proteasome inhibitors, as expected from their non-covalent chemistry.Dialysis of a preincubated mixture of c-20S and PKS21004 fully restoredof β5c activity (FIG. 1A). Kinetic analysis indicated that PKS21004 is anoncompetitive inhibitor of β5i and β5c with respect with theirsubstrates, respectively. With increasing concentration of PKS21004,V_(max) decreased and K_(M) remained constant in the case of β5cinhibition, and V_(max) and K_(M) both decreased in the case of β5iinhibition (FIGS. 1B-E), indicating that inhibition of β5i and β5c byPKS21004 are of mixed type noncompetitive with α_(c-20S)≈0.57 andα_(i-20S)≈0.28, indicating that PKS21004 binds more tightly to the β5cand β5i with substrate bound than without substrate, respectively.Decreasing V_(max)/K_(M) with increasing PKS21004 concentration alsosuggests that PKS21004 is not an uncompetitive inhibitor of either 20S(Copeland R. A., Evaluation of Enzyme Inhibitors in Drug Discovery: AGuide for Medicinal Chemists and Pharmacologists, 2d Ed., John Wiley &Sons, Inc., Hoboken, N.J., pp. 1-538 (2013), which is herebyincorporated by reference in its entirety).

The foregoing features of these AsnEDA encouraged a further round of SARstudies (Table 5) based on varying the carboxylic acid at theethylenediamine, the N-cap at the Asn, and the side chain of the Asn,based on PKS21004. All compounds listed in the Table 5 were synthesizedas described in Examples 6-77. All final compounds were confirmed by NMRand HRMS. IC50s of all compounds against β5i and β5c (Table 5), β1i,β2i, β1c and β2c were determined following a reported method (Lin etal., “N,C-Capped Dipeptides With Selectivity for MycobacterialProteasome Over Human Proteasomes: Role of S3 and S1 Binding Pockets,” JAm Chem Soc. 135:9968-9971 (2013), which is hereby incorporated byreference in its entirety). All compounds were specific for the 35subunit; no inhibition of β1i, β1c, β2i or β2c was observed, noinhibition <33 μM. Comparing the ethylenediamine with amethyl-ethylenediamine (PKS21018) and a 1,3-propyldiamine (PKS21019)indicated that the ethylenediamine gave the greatest potency for β5i andselectivity over β5c. The [1,1′-biphenyl]-3-carboxamide of PKS21004 wasthen modified with the following substituents: 4′-fluoro (PKS21026),4′-cyano (PKS21028), 4-fluoro (PKS21196), 4,3′-difluoro (PKS21195) and4,2′-difluoro (PKS21187). The 4′-substitutions decreased potency, while4- and 4,3′-substitutions did not. Best, 4,2′-difluoro-(PKS21187)improved potency against β5i to IC50 0.015 μM and improved selectivityto ˜20-fold over inhibition of β5c.

Next, an Asp-′Bu substitution on PKS21277, an intermediate in thesynthesis of PKS21187, was investigated. PKS21277 was modestly potentagainst β5i with 36-fold selectivity over β5c. Replacing the Asp-^(t)Buwith Gly (PKS21276), Ala (PKS21280), or Asn (PKS21281) abolished theinhibitory activities against both β5i and 05c, suggesting thatAsp-^(t)Bu is critical for optimal binding to β5. Phenylpropionate wasthen replaced with tosyl on the N-cap of the Asn of PKS21187, yieldingPKS21221. IC50s were determined to be 2.4 nM against β5i and 70 nMagainst β5c, representing 30-fold selectivity. Again, replacing theAsp-^(t)Bu of PKS21221 with Gly (PKS21289), Ala (PKS21290) or Asn(PKS21288) eliminated inhibitory activity against both β5i and β5c.

To corroborate that the noncompetitive modality of inhibition was sharedamong this class of compounds, PKS21221 was tested against β5i andconfirmed the non-competitive mechanism and c was determined to be 0.26(FIG. 5), in agreement with that of PKS21004.

To determine if the class of inhibitors was cell penetrable, the B-celllymphoma line Karpas 1106P (Singh et al., “Immunoproteasomeβ5i-Selective Dipeptidomimetic Inhibitors,” Chem. Med. Chem.11(19):2127-2131 (2016), which is hereby incorporated by reference inits entirety) (expressing a high proportion of i-20S over c-20S) wastreated with PKS21221 before incubation with either (Ac-ANW)₂—R110 (aspecific substrate of β5i) or suc-LLVY-luciferin (a substrate of β5).IC50 values with both substrates were identical and indicatedcell-penetrating ability. Similarly, PKS21221 inhibited β5c activity inHepG2 hepatoma cells with an IC50 of 2.0 μM. No β5i activity wasdetected in HepG2 cells using (Ac-ANW)₂—R110 as substrate (FIG. 2A).Correlating to varying PKS21221's intracellular proteasome inhibition inimmune and regular cell lines, cytotoxicity of PKS21221 against multiplemyeloma cell lines MM1.S and 8226 (FIG. 2B and Table 3).

In summary, a novel chemotype of proteasome inhibitors thatnon-covalently and noncompetitively inhibit the chymotryptic 05 subunitsof the proteasomes was identified. AsnEDA analogues as the selectiveinhibitors of the β5i over the 35c were developed. This is the firstreported example of potent, noncovalent, noncompetitive, and selectiveβ5i inhibitors. Unlike the competitive inhibitors whose intracellularactivity is often diminished over time when substrates buildup,noncompetitive inhibitors, on the other hand, retain the inhibitoryactivity, and in this case of the β5i inhibition, the buildup ofsubstrate actually enhances the binding of the inhibitor, which maybroaden the therapeutic window for treatment of autoimmune andinflammatory disorders. The versatility of the AsnEDA chemotype forproteasome inhibitors will be further demonstrated in a companion paperdescribing the development of selective inhibitors for the Plasmodiumfalciparum: proteasome over human host proteasomes.

Although the invention has been described in detail, for the purpose ofillustration, it is understood that such detail is for that purpose andvariations can be made therein by those skilled in the art withoutdeparting from the spirit and scope of the invention which is defined bythe following claims.

What is claimed:
 1. A compound of Formula (I):

wherein R is H or C₁₋₆ alkyl R¹ is selected from the group consisting ofalkyl, alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclic andbicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclicheterocyclyl and bi-heterocyclyl, and monocyclic and bicyclicnon-aromatic heterocycle, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle can be optionallysubstituted from 1 to 3 times with a substituent selected independentlyat each occurrence thereof from the group consisting of halogen, cyano,—OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl, heteroaryl, non-aromaticheterocycle, and non-aromatic heterocycle substituted with =0; R² isindependently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl; R³ isselected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl; R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, andC₃₋₈ cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substitutedwith —CF₃; R⁵ is selected from the group consisting of alkyl, alkenyl,monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, andmonocyclic and bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl can be optionally substituted from 1 to 3times with a substituent selected independently at each occurrencethereof from the group consisting of halogen, cyano, —OH, —NO₂, —CF₃,—OC₁₋₆ alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclicand bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl; k is0 or 2; m is 1 or 2; n is 1, 2, or 3; and p is 1 or 2; or an oxidethereof, a pharmaceutically acceptable salt thereof, a solvate thereof,or a prodrug thereof.
 2. The compound according to claim 1, which hasthe Formula (Ia):


3. The compound according to claim 2, which has the Formula (Ib):


4. The compound according to claim 2, which has the Formula (Ic):


5. The compound according to claim 1, wherein alkyl is C₁₋₆ alkyl. 6.The compound according to claim 1, wherein alkenyl is C₂₋₆ alkenyl. 7.The compound according to claim 1, wherein R¹ is


8. The compound according to claim 1, wherein R¹ is selected from thegroup consisting of


9. The compound according to claim 1, wherein R² is selected from thegroup consisting of H, CH₃,


10. The compound according to claim 1, wherein R³ is selected from thegroup consisting of H,

and R is C₁₋₆ alkyl.
 11. The compound according to claim 1, wherein thecompound of Formula (I) is


12. The compound according to claim 1, wherein the compound of Formula(I) is selected from the group consisting of:


13. A method of treating cancer, immunologic disorders, autoimmunedisorders, neurodegenerative disorders, or inflammatory disorders in asubject or for providing immunosuppression for transplanted organs ortissues in a subject, said method comprising: administering to thesubject in need thereof a compound of the Formula (I):

wherein R is H or C₁₋₆ alkyl; R¹ is selected from the group consistingof alkyl, alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclicand bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclicheterocyclyl and bi-heterocyclyl, and monocyclic and bicyclicnon-aromatic heterocycle, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle can be optionallysubstituted from 1 to 3 times with a substituent selected independentlyat each occurrence thereof from the group consisting of halogen, cyano,—OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl, heteroaryl, non-aromaticheterocycle, and non-aromatic heterocycle substituted with ═O; R² isindependently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic monocyclic and bicyclic aryl, monocyclic and bicyclicheteroaryl, monocyclic and bicyclic heterocyclyl can be optionallysubstituted from 1 to 3 times with a substituent selected independentlyat each occurrence thereof from the group consisting of halogen, cyano,—OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, alkyl, alkenyl, monocyclic and bicyclicaryl, monocyclic and bicyclic heteroaryl, and monocyclic and bicyclicheterocyclyl; R³ is selected from the group consisting of H, —SO_(p)R⁵,—C(O)R⁵, —C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl,—C(O)(CH₂)_(k)Het, —C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl(Ar) and heteroaryl (Het) can be optionally substituted from 1 to 3times with a substituent selected independently at each occurrencethereof from halogen or C₁₋₆ alkyl; R⁴ is selected from the groupconsisting of H, C₁₋₆ alkyl, and C₃₋₈ cycloalkyl, wherein C₃₋₈cycloalkyl can be optionally substituted with —CF₃; R⁵ is selected fromthe group consisting of alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, and monocyclic and bicyclicheterocyclyl, wherein alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, and monocyclic and bicyclicheterocyclyl can be optionally substituted from 1 to 3 times with asubstituent selected independently at each occurrence thereof from thegroup consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆ alkyl,alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic and bicyclicheteroaryl, and monocyclic and bicyclic heterocyclyl; k is 0 or 2; m is1 or 2; n is 1, 2, or 3; and p is 1 or 2; or an oxide thereof, apharmaceutically acceptable salt thereof, a solvate thereof, or aprodrug thereof.
 14. The method of claim 13, wherein the compound ofFormula (I) has the Formula (Ia):


15. The method of claim 14, wherein the compound of Formula (I) has theFormula (Ib):


16. The method of claim 14, wherein the compound of Formula (I) has theFormula (Ic):


17. The method of claim 13, wherein R¹ is


18. The method of claim 13, wherein R¹ is selected from the groupconsisting of


19. The method of claim 13, wherein R² is selected from the groupconsisting of H, CH₃,


20. The method of claim 13, wherein R³ is selected from the groupconsisting of H,

and R is C₁₋₆ alkyl.
 21. The method of claim 13, wherein the compound ofFormula (I) is


22. The method of claim 13, wherein the compound of Formula (I) isselected from the group consisting of:


23. The method of claim 13, wherein an autoimmune disorder is treated,said autoimmune disorder being selected from the group consisting ofarthritis, colitis, multiple sclerosis, lupus, systemic sclerosis, andsjögren syndrome.
 24. The method of claim 13, wherein immunosuppressionis provided for transplanted organs or tissues, said immunosuppressionbeing used to prevent transplant rejection and graft-verse-host disease.25. The method of claim 13, wherein an inflammatory disorder is treated,said inflammatory disorder being Crohn's disease and ulcerative colitis.26. The method of claim 13, wherein cancer is treated, said cancer beingselected from the group consisting of multiple myeloma, lymphoma, andother hematological cancers.
 27. The method according to claim 13,wherein the said administering is carried out orally, topically,transdermally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, by intranasal instillation, byintracavitary or intravesical instillation, intraocularly,intraarterially, intralesionally, or by application to mucous membranes.28. A pharmaceutical composition comprising a therapeutically effectiveamount of the compound according to claim 1 and a pharmaceuticallyacceptable carrier.
 29. A method of inhibiting chymotryptic β5i in acell or a tissue, said method comprising: providing a compound ofFormula (I):

wherein R is H or C₁₋₆ alkyl; R¹ is selected from the group consistingof alkyl, alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclicand bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclicheterocyclyl and bi-heterocyclyl, and monocyclic and bicyclicnon-aromatic heterocycle, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle can be optionallysubstituted from 1 to 3 times with a substituent selected independentlyat each occurrence thereof from the group consisting of halogen, cyano,—OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl, heteroaryl, non-aromaticheterocycle, and non-aromatic heterocycle substituted with =0; R² isindependently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl; R³ isselected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl; R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, andC₃₋₈ cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substitutedwith —CF₃; R⁵ is selected from the group consisting of alkyl, alkenyl,monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, andmonocyclic and bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl can be optionally substituted from 1 to 3times with a substituent selected independently at each occurrencethereof from the group consisting of halogen, cyano, —OH, —NO₂, —CF₃,—OC₁₋₆ alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclicand bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl; k is0 or 2; m is 1 or 2; n is 1, 2, or 3; p is 1 or 2; and contacting a cellor tissue with the compound under conditions effective to inhibitchymotryptic β5i.
 30. The method of claim 29, wherein the compound ofFormula (I) has the Formula (Ia):


31. The method of claim 30, wherein the compound of Formula (I) has theFormula (Ib):


32. The method of claim 30, wherein the compound of Formula (I) has theFormula (Ic):


33. The method of claim 29, wherein R¹ is


34. The method of claim 29, wherein R¹ is selected from the groupconsisting of


35. The method of claim 29, wherein R² is selected from the groupconsisting of H, CH₃,


36. The method of claim 29, wherein R³ is selected from the groupconsisting of H,

and R is C₁₋₆ alkyl.
 37. The method of claim 29, wherein the compound ofFormula (I) is


38. The method of claim 29, wherein the compound of Formula (I) isselected from the group consisting of:


39. A method of treating infectious disease in a subject, said methodcomprising: administering to the subject in need thereof a compound ofthe Formula (I):

wherein R is H or C₁₋₆ alkyl; R¹ is selected from the group consistingof alkyl, alkenyl, monocyclic and bicyclic aryl, biphenyl, monocyclicand bicyclic heteroaryl and bi-heteroaryl, monocyclic and bicyclicheterocyclyl and bi-heterocyclyl, and monocyclic and bicyclicnon-aromatic heterocycle, wherein alkyl, alkenyl, monocyclic andbicyclic aryl, biphenyl, monocyclic and bicyclic heteroaryl andbi-heteroaryl, monocyclic and bicyclic heterocyclyl and bi-heterocyclyl,and monocyclic and bicyclic non-aromatic heterocycle can be optionallysubstituted from 1 to 3 times with a substituent selected independentlyat each occurrence thereof from the group consisting of halogen, cyano,—OH, —NO₂, —CF₃, —OC₁₋₆ alkyl, aryl, heteroaryl, non-aromaticheterocycle, and non-aromatic heterocycle substituted with ═O; R² isindependently selected at each occurrence thereof from the groupconsisting of H, alkyl, alkenyl, monocyclic and bicyclic aryl,monocyclic and bicyclic heteroaryl, monocyclic and bicyclicheterocyclyl, and —(CH₂)_(m)C(O)NHR⁴, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, monocyclic andbicyclic heterocyclyl can be optionally substituted from 1 to 3 timeswith a substituent selected independently at each occurrence thereoffrom the group consisting of halogen, cyano, —OH, —NO₂, —CF₃, —OC₁₋₆alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclic andbicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl; R³ isselected from the group consisting of H, —SO_(p)R⁵, —C(O)R⁵,—C(O)(CH₂)_(k)Ar, —SO₂Ar, —SO₂C₃₋₈ cycloalkyl, —C(O)(CH₂)_(k)Het,—C(O)C₁₋₆ alkyl, and —C(O)OC₁₋₆ alkyl, wherein aryl (Ar) and heteroaryl(Het) can be optionally substituted from 1 to 3 times with a substituentselected independently at each occurrence thereof from halogen or C₁₋₆alkyl; R⁴ is selected from the group consisting of H, C₁₋₆ alkyl, andC₃₋₈ cycloalkyl, wherein C₃₋₈ cycloalkyl can be optionally substitutedwith —CF₃; R⁵ is selected from the group consisting of alkyl, alkenyl,monocyclic and bicyclic aryl, monocyclic and bicyclic heteroaryl, andmonocyclic and bicyclic heterocyclyl, wherein alkyl, alkenyl, monocyclicand bicyclic aryl, monocyclic and bicyclic heteroaryl, and monocyclicand bicyclic heterocyclyl can be optionally substituted from 1 to 3times with a substituent selected independently at each occurrencethereof from the group consisting of halogen, cyano, —OH, —NO₂, —CF₃,—OC₁₋₆ alkyl, alkyl, alkenyl, monocyclic and bicyclic aryl, monocyclicand bicyclic heteroaryl, and monocyclic and bicyclic heterocyclyl; k is0 or 2; m is 1 or 2; n is 1, 2, or 3; and p is 1 or 2; or an oxidethereof, a pharmaceutically acceptable salt thereof, a solvate thereof,or a prodrug thereof.
 40. The method of claim 39, wherein the compoundof Formula (I) has the Formula (Ia):


41. The method of claim 40, wherein the compound of Formula (I) has theFormula (Ib):


42. The method of claim 40, wherein the compound of Formula (I) has theFormula (Ic):


43. The method of claim 39, wherein R¹ is selected from the groupconsisting of


44. The method of claim 39, wherein R² is selected from the groupconsisting of H, CH₃,


45. The method of claim 39, wherein R³ is selected from the groupconsisting of H,

and R is C₁₋₆ alkyl.
 46. The method of claim 39, wherein the compound ofFormula (I) is selected from the group consisting of:


47. The method according to claim 39, wherein the said administering iscarried out orally, topically, transdermally, parenterally,subcutaneously, intravenously, intramuscularly, intraperitoneally, byintranasal instillation, by intracavitary or intravesical instillation,intraocularly, intraarterially, intralesionally, and by application tomucous membranes.
 48. The method according to claim 39, wherein theinfectious disease is caused by bacterial, viral, parasitic, and fungalinfectious agents.
 49. The method according to claim 39, wherein theinfectious disease is caused by a bacteria selected from the groupconsisting of Escherichia coli, Salmonella, Shigella, Klebsiella,Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis,Mycobacterium avium-intracellulare, Yersinia, Francisella, Pasteurella,Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcusaureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria,Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea,Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis,Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponemapalladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsialpathogens, Nocardia, and Actinomycetes.
 50. The method according toclaim 39, wherein the infectious disease is caused by a fungalinfectious agent selected from the group consisting of Cryptococcusneoformans, Blastomyces dermatitidis, Histoplasma capsulatum,Coccidioides immitis, Paracoccicioides brasiliensis, Candida albicans,Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrix schenckii,Chromomycosis, and Maduromycosis.
 51. The method according to claim 39,wherein the infectious disease is caused by a viral infectious agentselected from the group consisting of human immunodeficiency virus,human T-cell lymphocytotrophic virus, hepatitis viruses, Epstein-BarrVirus, cytomegalovirus, human papillomaviruses, orthomyxo viruses,paramyxo viruses, adenoviruses, corona viruses, rhabdo viruses, polioviruses, toga viruses, bunya viruses, arena viruses, rubella viruses,and reo viruses.
 52. The method according to claim 39, wherein theinfectious disease is caused by a parasitic infectious agent selectedfrom the group consisting of Plasmodium falciparum, Plasmodium malaria,Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania,Trypanosoma spp., Schistosoma spp., Entamoeba histolytica,Cryptosporidium, Giardia spp., Trichimonas spp., Balatidium coli,Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, Ascarislumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes,Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necatoramericanis.
 53. The method according to claim 39, wherein the infectiousdisease is malaria.